Development of a Novel Anti-Infectivity Platform for the Treatment of Neglected Tropical and Infectious Diseases Andrea Marie Binnebose Iowa State University

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2018 Development of a novel anti-infectivity platform for the treatment of neglected tropical and infectious diseases Andrea Marie Binnebose Iowa State University

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Development of a novel anti-infectivity platform for the treatment of neglected tropical and infectious diseases

Andrea M. Binnebose

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Microbiology

Program of Study Committee: Bryan H. Bellaire Major Professor Balaji Narasimhan Jesse Hostetter Michael J. Wannemuehler Richard Martin

The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred.

Iowa State University

Ames, Iowa

2018

DEDICATION

I dedicate this dissertation to the most amazing and influential men in my life.

When this journey began so many years ago, I had not realized how quickly my boys would grow. You have become strong and healthy young men and I feel like I have missed so many of your firsts.You have made me stronger, better and more fulfilled than

I could have ever imagined. This dissertation is the culmination of our journey towards a

Ph.D. which was just like climbing an insurmountable mountain. Together you have walked hand in hand, step by step with me through all the hardships, missed pickup times, just give me ten more minutes, and I have to no idea what you are talking about mom, but that is ok tell me. I love you

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TABLE OF CONTENTS

LIST OF FIGURES ...... v

LIST OF TABLES ...... vii

ACKNOWLEDGMENTS ...... x

ABSTRACT ...... xii

CHAPTER 1 . INTRODUCTION ...... 1

CHAPTER 2 . LITERATURE REVIEW ...... 3 Drug delivery systems ...... 3 Infectious diseases targeted for nanotherapeutic development ...... 6 Lymphatic filariasis ...... 6 Mycobacterium ...... 14 References ...... 31

CHAPTER 3 . POLYANHYDRIDE NANOPARTICLE DELIVERY PLATFORM DRAMATICALLY ENHANCES KILLING OF FILARIAL WORMS ...... 48 Abstract ...... 48 Introduction ...... 50 Materials and methods ...... 53 Results ...... 60 Discussion ...... 70 Conclusions ...... 75 Acknowledgements ...... 76 References ...... 79

CHAPTER 4 . NANOPARTICLE-BASED DRUG DELIVERY INCREASED SYNERGISM OF FIRST-LINE ANTI-FILARIAL DRUG ACTIVITY AGAINST BRUGIA PAHANGI ...... 84 Abstract ...... 84 Introduction ...... 86 Materials and methods ...... 91 iv

Results ...... 97 Confocal Microscopy ...... 105 Discussion ...... 108 References ...... 112

CHAPTER 5 . THE EFFECTIVENESS OF A NANO-BASED DELIVERY PLATFORM AGAINST MYCOBACTERIUM MARINUM ...... 118 Abstract ...... 118 Introduction ...... 119 Materials and methods ...... 122 Results ...... 127 Discussion ...... 136 Conclusion ...... 140 References ...... 140

CHAPTER 6 . AMPHIPHILIC POLYANHYDRIDE NANOPARTICLE DRUG DELIVERY PLATFORM EFFECTIVELY KILLS MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS...... 145 Abstract ...... 145 Introduction ...... 146 Materials and methods ...... 148 Results ...... 153 References ...... 160

CHAPTER 7 . NANOBASED DRUG DELIVERY ENHANCES THE ACTIVITY OF ANTIBIOTIC COCKTAIL AGAINST MULTI-DRUG RESISTANT MYCOBACTERIUM ...... 164 Abstract ...... 164 Introduction ...... 165 Materials and methods ...... 168 Results ...... 177 Discussion ...... 189 Conclusion ...... 192 References ...... 193

CHAPTER 8 . CONCLUSIONS AND FUTURE WORK ...... 197 References ...... 202 v

LIST OF FIGURES

Figure 2-1 Structures of poly(CPH) and poly(CPTEG)...... 5

Figure 2-2 Schematic representation of the life cycle of lymphatic filariasis ...... 8

Figure 2-3 Route of infection and development of primary disease by MTB ...... 18

Figure 2-4 Granuloma comprised of aggregate immune cells ...... 27

Figure 3-1 Release kinetics of ivermectin (IVM) and doxycycline (DOX) ...... 61

Figure 3-2 Table outlining survival of B. malayi females...... 64

Figure 3-3 Table outlining survival of B. malayi males ...... 66

Figure 3-4 The motility of B. malayi MF ...... 67

Figure 3-5 Confocal microscopy of female B. malayi with nanoparticles...... 69

Figure 3-6 Synergism of ivermectin (IVM) and doxycycline (DOX) ...... 77

Figure 3-7 Enzymatic reduction of MTT reagent by adult B. malayi females...... 78

Figure 4-1 Comparison of Polyanhydride nanoparticle formulations on B. pahangi females...... 99

Figure 4-2 Effects of antibiotics on B. pahangi female worms...... 102

Figure 4-3 Effects of second-line antibiotics on B. pahangi female worms...... 103

Figure 4-4 Effects of antibiotics on B. pahangi MF motility...... 106

Figure 4-5 Confocal microscopy of female B. pahangi with nanoparticles and soluble controls...... 107

Figure 4-6 Confocal microscopy of female B. pahangi with nanoparticles...... 108

Figure 5-1 M. marinum drug sensitivity profiles ...... 129

Figure 5-2 The killing of M. marinum is enhanced by using nanoparticles...... 131 vi

Figure 5-3 The effectiveness of four drug cocktail against M. marinum...... 132

Figure 5-4 The killing of intracellular M. marinum ...... 133

Figure 5-5 Intracellular targeting of NP in M. marinum infected cells...... 135

Figure 5-6 Internalization and intracellular viability of mycobacteria ...... 136

Figure 5-7 Intracellular growth of M. marinum-GFP ...... 139

Figure 6-1 Antimicrobial activity of RIF/PZA NP against MAP...... 154

Figure 6-2 Antimicrobial activity of INH/EMB NP against MAP...... 155

Figure 6-3 Antimicrobial activity of RIP/PZA/INH/EMB NP against MAP...... 156

Figure 6-4 Internalization and intracellular viability...... 157

Figure 6-5 Intracellular viability MAP within murine macrohpages...... 158

Figure 6-6 Intracellular viability MAP within murine macrohpages following treatment with RIF/PZA/INH/EMB CPH:SA NP ...... 159

Figure 7-1 Antimicrobial activity of NP-RIPE against XDR-TB M. tuberculosis ...... 178

Figure 7-2 The killing of M. tuberculosis MDR-TB ...... 179

Figure 7-3 Killing of M. tuberculosis H37Rv ...... 180

Figure 7-4 Intracellular viability of M. tuberculosis ...... 183

Figure 7-5 The use of the drugs against H37Rv in vivo...... 184

Figure 7-6 The use of drugs against MDR-TB in vivo...... 185

Figure 7-7 The use of the drugs against XDR-TB in vivo...... 186

Figure 7-8 C3H mice weights ...... 187

Figure 7-9 The in vitro release and encapsulation profiles of pyrazinamide rifampicin, and isoniazid...... 188

Figure 7-10 Inhibitory activity of the released drug against M. marinum...... 189

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LIST OF TABLES

Table 2-1 Lymphedema Stage and Its Characteristics ...... 7

Table 2-2 Recommended dosage of Antifilarial ...... 10

Table 2-3 The classification of the drugs used for the treatment of drug-resistant tuberculosis ...... 20

Table 2-4 Recommended dosage of first-line antituberculous drugs ...... 28

Table 7-1 Level of DNA relatedness among strains of Mycobacterium species ...... 172

Table 7-2 Mycobacterial drug susceptibility ...... 173

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Abbreviations

ADME An acronym for absorption, distribution, metabolism and excretion AIDS Acquired immune deficiency syndrome ALB Albendazole ATP Adenosine triphosphate BCG Bacillus Calmette-Guérin BSA Bovine serum albumin CFU Colony Forming Units DEC Diethylcarbamazine DMSO Dimethyl sulfoxyde DOX Doxycycline EMB Ethambutol IC50 Half maximum inhibitory concentration IFN-γ Interferon-gamma IL Interleukin INH Isoniazid IVM Ivermectin LF Lymphatic Filariasis LSC Laser Scanning Confocal MAP Mycobacterium avium subspecies paratuberculosis MDR-TB Multi-drug Resistant Mycobacterium tuberculosis MIC Minimum inhibitory concentration MM Mycobacterium marinum MTB Mycobacterium tuberculosis NLC Nanostructured lipid carrier NTM Nontuberculosis Mycobacterium OD Optical density ix

PANs Amphiphilic polyanhydride nanoparticles PLA Polylactic acid PLGA Poly-D-L-lactide-co-glycolide PZA Pyrazinamide RIF Rifampicin RIPE Rifampicin, Isoniazid, Pyrazinamide, Ethambutol TDR-TB Totally-Drug Resistant Mycobacterium tuberculosis TNF-α Tumor necrosis factor α WHO World Health Organization WT-TB Wild type Mycobacterium tuberculosis XDR-TB Extensively-drug Resistant Mycobacterium tuberculosis

ACKNOWLEDGMENTS

First and foremost, I would like to thank God Almighty for providing me the opportunity, knowledge, perseverance, and ability to undertake this journey and complete it.

To my mentor Dr. Bryan H. Bellaire who has provided numerous research opportunities, for his mentorship, friendship, and for sharing his time, scientific knowledge and wisdom over the last five years. He has provided mentorship that inspires continual self-improvement resulting in a substantial independent and scientific competence, and for that, I am genuinely grateful. I would also like to thank my committee members; Dr. Balaji Nara- simhan, Dr. Mike Wannemuehler, Dr. Richard J. Martin and Dr. Jesse Hostetter, for there continual support, and guidance.

I would like to give a special thanks to Dr. Narasimhan and his lab members

Shannon, Adam, and Sean. A special thanks to Adam for making me countless batches of nanoparticles and working with me on the San Francisco trip. I will never forget that trip!

Thanks to Dr. Saurabh Verma who helped with the filarial dissections. To my lab mate and partner in crime Nathan, for helping me in the BSL3 with the animal experiments and for letting me talk his ear off without complaining. I also would like to thank an exceptional mentor Dr. Susan Bornstein-Forst. She was the original driving force who in- spired and made me believe that I could pursue a Ph.D. when others said I could not.

I owe a very special thank you to my family who have supported and listened to me through every accomplishment and frustration that this arduous journey has had to offer. To my father, thank you for having a huge heart and spending so many of your weeks and months here in Iowa helping with the kids and supporting me. To my beloved hus- band, thank you for taking the chance on an unknown, for being my rock through all the xi trials and frustrations that occurred. Lastly, I would like to thank my three sons, what can

I say but we did it! You went willingly on an adventure, putting your complete faith and love in me, you have been there when I cried and laughed providing me with the inspiration and strength to keep going forward. You were my inspiration! Without the guidance and support from these special people, this accomplishment would not have been possible.

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ABSTRACT

This dissertation focuses on the design of novel strategies for the prevention and treatment of neglected tropical and infectious diseases using polyanhydride nanoparticles as a drug delivery platform. The overall goal of this research is to design efficacious drug delivery vehicles that are effective against the filarial nematode Brugia malayi and B. pahangi and intracellular bacteria Mycobacterium tuberculosis, M. paratuberculosis, and

M. marinum. The primary goal is to use the polyanhydride nanoparticles to deliver high enough microenvironmental concentrations to cause the death of the organism, thereby reducing the mortality, morbidity, and transmission of the disease. While a wide array of vaccine delivery vehicles have been investigated the majority of the research has focused on the use of polyesters and polyanhydride. This research presented shows the benefits of biodegradable polyanhydride as an effective drug delivery platform using the polymers and copolymers of various chemistries.

We demonstrate the use of a polyanhydride nanoparticle-based platform for the co-delivery of the antibiotic doxycycline with multiple antiparasitic drugs, (ivermectin, diethylcarbamazine, and albendazole) to reduce microfilariae burden and to rapidly kill adult worms. By encapsulating two of these drugs, ivermectin, and doxycycline, into biodegradable polyanhydride nanoparticles, we report the ability to effectively kill adult B. malayi and B. pahangi worms with up to a 4,000-fold reduction in the amount of drug used. Additionally, we significantly reduced the average time to death of the macrofilaria

(five-fold) when the anti-filarial drug cocktail was delivered within polyanhydride nanoparticles. xiii

Knowledge gained from filarial studies was then applied to kill the intracellular bacteria Mycobacterium, we sought to examine the effectiveness of the first line antituberculous rifampicin, isoniazid, pyrazinamide, and ethambutol, encapsulated into various polymer and copolymer combinations in vitro. Initial experiments focused on particle internalization, co-localization, and reduced macrophage bacterial population. Subse- quently, gained knowledge from the in vitro effectiveness was then applied to systemic acute M. tuberculosis in vivo infection focusing on multi-drug resistance and extensively drug-resistant strains. We observed through short course chemotherapy utilizing the nanoparticle-based drug delivery platform 20:80 CPTEG:CPH, we were able to reduce the prevalence of a persister bacterial population significantly. In summary, the studies described herein support the rational design and use of a polyanhydride nanoparticle drug delivery platform to reduce both the course of treatment and the amount of drug needed to treat neglected infectious disease effectively. 1

CHAPTER 1 . INTRODUCTION

From the first discovery of penicillin in 1928 to the first successful therapeutic use in 1941, antibiotics have had a profound impact on survival and reducing the overall microbial burden on humans and animals. Despite their success, overuse and misinfor- mation have led to the development of antimicrobial resistance. Our early understanding of the interactions of antibiotics with microorganisms and how they effectively overcome our most potent chemotherapy was, in fact, rudimentary at best. Because of our overconfidence in the abilities of these chemotherapies, without understanding the pathogenesis and specific virulence mechanism of these microorganisms, we are faced with substantial infectious disease challenges.

This dissertation is presented in two major sections: The first is actionable research that focuses on overcoming the challenges associated with neglected tropical diseases such as Lymphatic Filariasis. The research will focus on strategies for the development of a biodegradable polyanhydride nanoparticle-based platform for the co- delivery of small molecules, to reduce the microfilariae burden and rapidly kill adult worms. The second section will focus on the development of a polyanhydride nanoparticle platform for the delivery of a four-drug cocktail against multi-drug resistant

Mycobacterium species through reducing the bacterial burden in vivo.

Antimicrobial therapies directed against M. tuberculosis must overcome numerous difficulties to be successful at reducing morbidity and mortality of the infection. The difficulties challenging M. tuberculosis infections results from the layered seclusion of the organism within 1) a walled structure (granuloma) within the lungs 2) within the 2 intracellular microenvironment of monocytes and macrophages 3) a thick hydrophobic mycolic acid protective outer layer which reduces permeability to antibiotics. Within the monocyte and macrophage host cells, the bacteria are not destroyed and establish an intracellular niche that further impedes antibiotic delivery. The studies presented will out- line a methodical experimental approach that addresses these challenges in the treatment of mycobacteria. We will review and address the challenges the current chemotherapies possess, with a strategic approach in developing a novel antimicrobial drug delivery platform utilizing polyanhydride nanoparticle to address the presences of multi-drug resistant

M. tuberculosis. It is anticipated that these studies will help guide the next decade of antimicrobial research and development.

CHAPTER 2 . LITERATURE REVIEW

Drug delivery systems

Over the last ten years, new and innovative technologies have spurred the development of biodegradable nanoparticles for use as a drug delivery platform. There are currently four classifications of delivery systems [1]. These new and FDA approved platforms includes various particle technologies and vary in size from 10-1000 nm. As a technology, nanoparticles are capable of delivering a variety of payloads by implement- ing heterogeneity in materials and methods [2]. The payloads can be both hydrophilic and hydrophobic, and the specific application is primary in the development. Examples of payloads include small molecules, proteins, peptides, and nucleic acids. Because of the volatile nature of these payloads, the particle system must provide increased encapsulation efficiency, controlled release mechanisms, tissue-specific active targeting and improvement of therapeutic indexes. There is a need to develop better characterization tech- niques for quality control, consistency in synthesis, reduction in development and manu- facturing costs to facilitate the successful translation of theoretic applications of nano- technologies into approved clinical therapies. A critical benefit of nanoparticle-based therapies is their ability to be internalized through phagocytosis, which presents a particular advantage in situations where standard internalization mechanisims would be challenging for a given molecule [3].

Solid Lipid Nanoparticles and Microparticles

Lipid nanoparticles are a new generation of colloidal drug carrier systems that are solid at basal and room temperature. Solid lipid nanoparticles are comprised of up to

2.5% of physiological lipids and have not shown toxicity issues to date. They typically contain a hydrophobic core with a phospholipid coating which provides increased drug loading and the ability to encapsulate bioactive and hydrophilic compounds [4]. These nanoparticles, in general, possess adequate stability for periods of more than a year and are highly stable. However, due to the homogenization processes which requires excess heat and excessive handling, degradation occurs on the active components during the encapsulation process [5]. The administration is primarily through the dermal route although, intravenous injection has been attempted consistency in particle size is a challenge [3].

Emulsion-based delivery systems

Emulsion-based delivery systems are becoming increasingly prevalent in encapsulating lipophilic components. The emulsion delivery system comes in three different forms and is composed of an oil and water phase[6]. Homogination of the aqueous phase with the oil phase solubilizes the lipophilic component and requires thermodynamic sta- bilization through combinations of surfactant [7, 8], resulting emulsions of micro >100 nm or nano <100 nm. Emulsions systems protect enzymatic hydrolysis [9] and stabilize the bioavailability of the lyophilic components [10]. As a drug delivery system, they enhance the therapeutic efficacy of the drug, minimize the adverse effect and toxic reactions, a larger surface area providing greater absorption and can be formulated into 5 foams, creams, liquids, and sprays[11]. Use of the emulsion systems allows for a variety of administration applications and it is one of a most promising delivery system for poorly soluble drugs

[12]. Poly(CPH)

Polymeric nanoparticles

Polyesters Poly(CPTEG)

Individual Polyester, or in combination with other polymers are the most investigated biodegradable polymer and have been widely adapted for the use as drug carrier NPs. Poly (lactic acid) (PLA) and poly(lactic-co-glycolic acids) are the most versatile of the polyesters with an array of different Figure 2-1 Structures of poly(CPH) and poly(CPTEG). MWs, compositions and variable degradation rate [13]. Also shown is a scanning photomicrograph of drug-loaded CPTEG:CPH NPs. Scale: 1 µm. Polyesters are among the most popular choices due to their high biocompatibility and biodegrade property despite generation of acidic by-products which directly interferes with encapsulation stability. PLGA polymer formulations are compatible with injectable delivery of peptides, therapeutic proteins, hormones, and antibiotics.[14]

Polyanhydride

Polyanhydride nanoparticles have been studied extensively as carriers for drugs and vaccines for over 30 years [15-19]. These NPs are FDA approved, as evidenced by the

Glidal polyanhydride wafer consisting of 1,3-bis(p-carboxyphenoxy)propane (CPP) and sebacic acid (SA) [20]. These biodegradable materials degrade into dicarboxylic acids upon scission of the anhydride bond, rendering them highly biocompatible. These materials 6 are hydrophobic, and this confers a surface erosion mechanism to devices made of polyanhydrides [21, 22]. The advantage of such a surface erosion mechanism is that payloads can be released in a sustained and predictable manner, providing a so-called zero order release profile, in which the release rate of the payload is constant. Such release profiles enable single-dose therapies, maximize the time over which the in vivo drug concentration is maintained within the therapeutic window, and enhance patient compliance.

Infectious diseases targeted for nanotherapeutic development

Lymphatic filariasis

Filarial parasites belonging to the family Onchocercidae remain a significant global burden, endemic in over 80 countries worldwide, particularly India and sub-Sa- haran Africa [23]. Currently, there are upwards of 120 million individuals at risk [23-25] with an average of 38.5 million people disfigured and incapacitated by the disease. The primary human diseases caused by filariae are Lymphatic Filariasis (LF), River Blindness

(RB), and Loiasis. The parasitic helminth Wuchereria bancrofti, Brugia malayi, and B. timori are the causative agents of Lymphatic Filariasis (LF). Of the Onchocercidae, On- chocerca volvulus causes RB. While efforts to mitigate the effect of these diseases have been successful in some regions, current medications are insufficient to reach elimination by 2020 [26-28]. Most cases of the disease have little to no symptom presentation. Some individuals, however, develop a syndrome called elephantiasis, which is characterized by fever, lymphangitis, hydrocele, and lymphoedema, and is one of the most common causes of arthropod mediated disability [28, 29].

Table 2-1 Lymphedema Stage and Its Characteristics

Lymphedema Stage and Its Characteristics (Dreyer et al., 2002).

Lymphedema Stage Characteristics

Stage 1. Swelling goes away overnight

Stage 2 Swelling does not go away overnight

Stage 3 Shallow skin folds

Stage 4 Knobs

Stage 5 Deep skins folds

Stage 6. Mossy lesions

Stage 7 Unable to care for self or perform daily activities

Morbidity associated with infection stems from both the persistence of the worm in the skin and the lymphatics that creates scarring and fluid accumulation and the larval form of LF microfilariae (MF) causing debilitating chronic dermatitis due to irritation of MF antigen [30].

Lifecycle

The unique parasitic life cycle begins (Figure 1) when the larval microfilariae

(MF) are picked up during a blood meal from an infected host by the insect vector and remain in the thoracic muscles of the mosquitoes. The principal mosquito vectors include

Mansonia, Anopheles, and Aedes mosquitoes. The microfilariae (L1) undergoes two larval molting’s stages L2, and the infective L3 stage, this takes around 10-14 days [31].

When the infected mosquito feeds on another human host, the infective larvae (L3) are deposited within host tissue at the bite site and migrate to the lymphatics.

Figure 2-2 Schematic representation of the life cycle of lymphatic filariasis, showing parasite development in the human host and vector.

One of the unique characteristics of the L3 stage is its ability to modulate and suppress the host immune response via secretion of proteins and microRNAs [32, 33]. These proteins and microRNAs allow for the initial suppression of the innate immune response 9 which gives the opportunity for the L3 stage to mature into the adult worms (macrofilaria). The host’s immune response to the microfilariae involves many different cell populations, which include granulocytes such as eosinophils, mast cells and polymorphonu- clear leukocytes (PMNs), as well as monocytes. These immune cell populations can recognize, trap and kill the blood circulation microfilaria (L1) stage in the human filarial parasitic nematode Brugia malayi [34, 35]. For the majority of the day, microfilariae sequester within the lungs of the host and only appear in the peripheral blood circulation for a few hours each night, which coincides with maximal mosquito feeding [36].

Endosymbiotic bacteria

Resident within W. bancrofti and B. malayi parasites at all stages of life are the endosymbiotic bacteria, Wolbachia. These obligate intracellular bacteria are members of the Rickettsialaes order of α-proteobacteria [37] that also contain the mammalian pathogens in the genera Rickettsia, Brucella, Bartonella, Ehrlichia and Anaplasma [38]. Typi- cally, Wolbachia is present within hypodermal cells of the lateral cords of both sexes.

However, vertical transmission of the bacteria to MF is facilitated by the presence of bacteria in the ovaries, oocytes, and embryos during development in females [39].

Wolbachia, are a vital component of larval development and adult-worm fertility and viability.

Host response

A single female macrofilaria can produce over a billion MF during this time [40,

41]. The host’s immune response to the microfilariae involves many different cell populations, which include granulocytes such as eosinophils, mast cells and polymorphonu- 10 clear leukocytes (PMNs), as well as monocytes. These immune cell populations can recognize, trap and kill the blood circulation microfilaria (L1) stage in the human filarial parasitic nematode Brugia malayi [34, 35]. For the majority of the day, microfilariae sequester within the lungs of the host and only appear in the peripheral blood circulation for a few hours each night, which coincides with maximal mosquito feeding [36].

Treatment

No vaccine is currently available; filariases are controlled through chemotherapy, although vector control and bed-nets are important. Current treatment protocols primarily focused on interrupting the spread of LF by targeting the microfilariae larvae with combinations of anti-helminthic medications. The drugs most frequently used to manage LF include albendazole (ABZ), a benzimidazole derivative; diethylcarbamazine (DEC), a derivative of piperazine; which is involved in modulation of the host immune response to be effective [42] and Ivermectin (IVM), which is believed to be a broad-spectrum antiparasitic agent [43-47]. Ivermectin has been shown in animal parasites to modulate the activity of PMNs as well as monocytes [35, 48].

Table 2-2 Recommended dosage of Antifilarial

Recommended doses of antifilarial for adults.

Drug Daily administration Administration Standard dose Maximal dose Standard dose Total maximal doses (mg) (mg/kg) (mg/kg) Ivermectin 200 150-200mg/kg 1-2 (IVM) 11

Table 2-2 continued Diethylcarbam- 100 6 mg/kg 12 azin (DEC) Albendazole 400 400 mg 1 (ABZ) Doxycycline 600 100-200 mg/kg 42 (DOX) for 42 -

These drug combinations are used primarily for long-term, community-directed treatments aimed at interrupting transmission. Some of the possible side effects of these drugs include fever, itching, oedema, arthritis, and lymphadenopathy [49]. These side effects are primarily due to the extensive paralysis and killing of the microfilariae. These medications have minimal activity against the causative organism, adult Wuchereria bancrofti, Brugia malayi and B. timori worms, and have thus far, failed to deliver a radical curative treatment. This failure is due to the unique composition of the outer cuticle and the specificity of infected tissue limits, and the permeability of these compounds

Research has highlighted the use of the above drugs with macrofilaricidal activity as a single therapy or combinations could improve the probability of a curative treatment.

This approach has reduced the reoccurrence in some countries by 46% over the last 13 years [25, 50]. These antimicrobials have been shown to reduce the number of resident

Wolbachia, although the exact symbiotic role within the nematode is not directly known.

Research has shown that the use of DOX can cause long-term sterilization and death of adult female worms in 6 to 12 months with a 3-6 week treatment course [51-54].

However, limitations associated with the use of the above drugs is four-fold: specific restrictions on patient age and health status due to cytotoxicity; the inability to reach 12 broad tissues where the adult filarial nematodes reside; as well as counter-indications in areas endemic for the Loa loa parasite; and poor patient compliance.

An alternative antibiotic which has been tested and has shown effectiveness against Rickettsiae is rifampicin, which is better tolerated in children and pregnant patients and has been shown to be effective against Wolbachia in Onchocerca parasites in vitro [55]. However, rifampicin is less effective than doxycycline in vivo [56, 57]. Addi- tional therapeutic options for doxycycline could be useful when combined with current antiparasitic drugs; however, combination therapies present difficulties for drugs that have different systemic half-lives, similar toxicity profiles, low aqueous solubility and reduced shelf life properties. These equilibrium solubility issues can be exasperated by the differing comprehensive drug administration programs that already suffer from poor patient compliance, and multi-year therapy regimes [58].

Ivermectin

Ivermectin, is a broadly used macrocyclic lactone endectocid used in veterinary medicine for the treatment or prevention of nematode infections. In humans, IVM is one of the three mainstay drugs in the LF global elimination program [59]. IVM is primarily a microfilaricidal, due to the fast MF clearance that is observed. The killing effectiveness against the adults is moderate and requires repeated treatment cycles over several years.

Despite the effectiveness, the exact mode of action remains elusive. We do understand that ivermectin binds to the nematodes glutamate-gated chloride channels (GluCls), interrupting the neurotransmission processes that regulates [60] pharyngeal muscle movements [61, 62]. This interruption leads to the channeling irreversibility opening; leading 13 to hyperpolarization and depolarization of the neuron [63, 64]. Leading to muscular paralysis and inhibition of pharyngeal pumping, which do not directly kill the worm [65].

Doxycycline

Doxycycline (α-6-deoxy-5-oxytetracycline), is a broad spectrum semi-synthetic isomer antibacterial derived from tetracycline [66]. The primary mode of action is to inhibit protein synthesis by binding to the 30S or 50S ribosomal RNA subunits thereby preventing access of aminoacyl tRNA to the acceptor site on the mRNA-ribosome complex. Because inhibition of the tRNA requires active transport through adenosine triphosphate (ATP) the doxycycline effectively penetrates the cytoplasmic membrane[67]. Re- search done by [68, 69] showed a < 80% effectiveness against long-term sterilizing activities of adult worms render it as a “disease-curing “. This disease curie is believed to be a direct result of doxycycline inducing growth retardation, embryostasis, and inhibition of

MF shedding [70, 71].

Albendazole

Albendazole, is a heterocyclic aromatic organic compound derived from benzimidazole. Albendazole selectively binds to the nematodes β-tubulin and inhibits the micro- tubule polymerization in a two-step process [61, 72, 73] resulting in the destruction of cell structure and death of the worm [74].

Diethlycarbamazine

Diethlycarbamazine, is a piperazine derivative, which has been shown to kill at all stages of the parasite life-cycle [75]. Like ivermectin the exact mechanism by which killing occurs remains to be elucidated, DEC is thought to sensitize them to phagocytosis 14 through targeting of the arachadonic acid, 5-lipoxygenase, and cyclooxygenase-1 pathways. Studies have suggested that DEC indirect, host-mediated mode of action along with anti-inflammatory effects during treatments [76, 77]. Such studies suggested that use of DEC inhibits inducible nitric oxide synthase (iNOS), nuclear transcription factor kappa

B (NF-kB) activation, a regulator of the proinflammatory genes such as TNF-α, and IL-1

[78]. In addition DEC has also been shown too inferred with the host arachidonic acid and nitric oxide metabolic pathways [78, 79].

Mycobacterium

Mycobacterium marinum

Mycobacterium marinum is a “slow-growing” nontuberculosis mycobacterium

(NTM) that is most commonly a waterborne pathogen responsible for tuberculosis-like infections in ectotherms and is an occasional opportunistic human pathogen. Marinum is the closest genetic relative of the M. tuberculosis complex and as such is the most reliable in testing for drug susceptibility. Nontuberculous mycobacteria are responsible for a multiplicity of different types of infections, including respiratory, cutaneous, and systemic

[80], of which many of the species of NTM are multidrug-resistant [81], including M. marinum. M. marinum is an atypical bacilli’s that are reported to be resistant to the antituberculosis medications isoniazid, pyrazinamide, and para-aminosalicylic acid and shows intermediate sensitivity to streptomycin [82, 83]. Unlike M. tuberculosis slow growth rate of 3-6 weeks, M. marinum grows within 1-2 weeks. This faster growth rate allows for a model that can be correlated to effective drug susceptibility in vitro. 15

Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of chronic granulomatous enteritis in ruminants called Johne's disease (JD) [84] as well as suspected in the human inflammatory bowel diseases Crohn’s (CD) and Ulcerative Co- litis (UC) [85-89]. MAP is a nontuberculosis mycobacterium, of the Mycobacterium avium complex (MAC) most often associated with the small intestinal lamina propria of ruminants that is eventually fatal in many cases [90, 91]. The infection in ruminants is most commonly transmitted through the fecal-oral route due to contaminated closed confinement, pasture, soil, and water, as well as vertical intrauterine transfer [92]. The effects of JD on the U .S. dairy industry accounts for upwards of $220 million per year, with 2 out of every three farms being seropositive [93]. Like most mycobacterium infection symptomology does not occur until many months after initial expose and treatment in ruminates is not suggested.

Source of human contamination is believed to be a direct result of bacterial shedding into drinking water, milk and in even meat [94-96]. A significant characteristic found in Mycobacterium spp. is the lipid-rich cell wall which in MAP is believed to provide needed protection during pasteurization [97].The occurrences of MAP being found in individual testing positive for CD has been shown to exceed 59% [88, 89] but testing is limited and can be unreliable [94]. Individuals testing seropositive for the MAP may undergo extensive extended drug therapy (<5 weeks) [98] using the standard antituberculous rifampicin, isoniazid, ethambutol, and pyrazinamide [99] as well as 16 clythromycin [100]. Treatment of infected individuals caused by MAC remains a challenge since these organisms are resistant to the majority of antituberculous drugs [99,

101].

Mycobacterium tuberculosis

The world health organization (WHO) has reported that in 2016, 6.3 million new cases of M. tuberculosis were reported and nearly 1.3 million individuals died as a result

[28]. M. tuberculosis is the ninth leading cause of death worldwide and the leading cause of death from a single infectious agent [28, 102]. Mycobacterium tuberculosis (MTB) is a non-spore forming, acid fast, non-motile rod shaped bacilli which is highly resistant to solvents. In human medicine the most significant group of mycobacteria are the members of the Mycobacterium tuberculosis complex (consisting of M. tuberculosis, M. bovis, M. africanum, M. microti, M. pinipendi and M. canettii. Human MTB is a highly contagious infectious disease predominantly caused by M. tuberculosis. It is a weak Gram-positive rod-shaped bacterium that has no flagellum, does not form spores nor produces toxins and has no capsule. Mycobacterium’s size varies from 1 to 4 μm in length, respectively. It has been found to affect nearly all organs within the human body, but the lung is of particular high incidence. It is a intracellular pathogen that establishes its infection prefer- entially to macrophages of the respiratory system where it can be contained and enters into a dormancy state [103, 104].

The primary means of transmission occurs by infective particles (Figure 1-3) of as little as 1 to 10 bacilli are required for infection. Inhaled bacilli are phagocytised by primary alveolar macrophages where they avoid fusion with lysosomes and posterior di- gestion in the protective intracellular environment [105]. The first evidence of infection 17 usually occurs within six to eight weeks, followed by the development of cellular immunity and subsequent lymphocyte and activated macrophage to the site of infection. Where the primary population of bacilli are eliminated without manifestation of symptoms. The infected phagocytic macrophages and lung parenchymal cells are also killed, which produces a characteristic solid caseous necrosis consisting of macrophages and giant cells, T cells, B cells, and fibroblasts, and these granulomas can prevail not only in the lung, but in other organs as well. When host immunity predominates, the granuloma type lesion can be contained, resulting in residual damage to the lungs. The sustained necrotic lesion expands collapsing into the bronchus, where a exsentive lung lesion can be formed, result in systemic bacterial dissemination into the blood stream and airway. However, more commonly, mycobacteria can remain dormant or develop into a latent form of infection within the host, which establishes a bacterial reservoir [99]. Around 20% of individuals infected present extra-pulonary MTB, which is caused by development and evolution of baccili disseminating into the blood vessels. Miliary tuberculosis is widespread dissemination through the blood stream, where it cn be found within lymph nodes, liver, spleen, bones and joints, heart, brain, genital-urinary system, meninges, peritoneum and skin [104]. Mycobacterium’s pathogenesis and virulence factors produces typical symptoms such as weakness, fever, weight loss, night sweat, chest pain, respiratory insuffi- ciency and excessive cough, whereas advanced pathology may also cause bleeding, which leads to haemoptysis. Many symptoms including tissue destruction are mediated as a result of the host´s immune response and not directly as a result of the bacteria itself. 18

Figure 2-3 Route of infection and development of primary disease by MTB

Latent tuberculosis infection is defined as an infection with M. tuberculosis in combination with a positive tuberculin skin test or an interferon- γ (INF- γ) release assay results concurrently without indication of active TB symptoms, progressive radiographic changes or microbiological evidence of replicating organisms [106, 107]. The terms dormancy, persistence and latency have been used interchangeably generally to refer to the non-replicating state of mycobacteria, tolerance to antituberculotics or an infection without active disease. Dormancy is a physiological state of arrested growth, persistence consists in the phenotypic transient tolerance to the antibacterial effects of drugs [108-110]. 19

Latent tuberculosis is recognized as a result of the interaction of the host’s immunity with

M. tuberculosis infection, forcing it into a dormant state in the infected alveolar tissue.

Bacteria do not undergo any metabolic process in response to the host immune response.

During this dormancy period latent TB is in a state of non-infectious disease and, therefore, does not represent a public health threat. The bacterial intracellular survival depends on the phagosome acidification in infected macrophages thus preventing the phagosome- lysosome fusion.

Drug resistance

The prevalence of resistance within individuals infected with MTB currently exceed 10% of the newly reported cases were found to be resistance to the first line drugs, rifampicin and an additional 5% of these cases reported multi-drug resistance (MDR-TB) strains. These strains are reported to be resistant to at least rifampicin (RIF) and isoniazid

(INH) [111-113]. Individuals that succumb to this disease could be prevented with appropriate treatment and diagnosis. The clinical features of tuberculosis (TB) infection include multistage processes of primary infection, latency, and reactivation. Years of efforts have been made to reduce the adverse treatment and develop an understanding of the pathogenesis of the organism [114-116]

Frequent failures of the treatment of tuberculosis (TB) are due to the insufficient dosing, incomplete treatment regimens caused by poor patient compliance, and latency profile of the bacilli [117, 118]. Initial treatment requires a two-step process; the first involves an “intensive phase” of a four-drug high dose cocktail containing the antibiotics rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB). The

“intensive phase” can only last for to two-month due to high toxicity issues with the 20 antibiotic therapy. Immediately following the “intensive phase” a longer “continuation phase” occurs with RIF and INH for up to two years [28, 119, 120]. This secondary treatment is necessary to eradicate the remaining Mycobacterium that has entered a dormant or latent phase. The need for such lengthy treatment reflects the unique nature of the latent population of mycobacterium that remains in a non-replicating persistent state within caseous lung granulomas [121]. These “persister” bacilli are genetically drug- susceptible but exhibit a phenotypic tolerance to currently available first-line drugs [122,

123].

Table 2-3 The classification of the drugs used for the treatment of drug-resistant tuberculosis The classification of the drugs used for the treatment of drug-resistant tuberculosis. The groups of drugs used for the treatment of drug-resistant tuberculosis Group/drug Recommendation/drug First-line oral antituberculosis agents (use all possible drugs) isoniazid Ethambutol rifampicin pyrazinamide rifabutin Injectable agents (use one because of similar targets) capreomycin streptomycin kanamycin (viomycin) amikacin (enviomycin) Fluoroquinolones (use one because of the same target) ofloxacin moxifloxacin levofloxacin (gatifloxacin) Second-line oral antituberculosis agents (use all possible drugs if necessary) ethionamide cycloserine 21

Table 2-3 continued prothionamide terizidone p-aminosalicylic acid

Antibiotics with an unclear role in the (use all necessary drugs to complete four- treatment of MDR-TB combination of antituberculotics) clofazimine clarithromycin amoxicillin/clavulanate high-dosed isoniazid linezolid thioacetazone imipenem/cilastatin

Although the incidence and mortality rates of TB have significantly declined over the last ten years, the current advances in TB control remain hindered by the increase in reports of drug-resistance [123, 124]. Multidrug-resistant TB (MDR-TB), is defined as resistant to at least two antibiotics such as rifampicin and isoniazid [112, 113]. Extensively drug-resistant TB (XDR-TB) is a phenotypically adapted MDR which shows resistant to isoniazid and rifampin, plus fluoroquinolones and at least one of the injectable second- line drugs [111, 125]. Furthermore, cases of totally drug-resistant tuberculosis (TDR-TB) have originated in Asia, Africa, India and Europe. In TDR-TB, the Mycobacterium is resistant to all available drug chemotherapy’s [102].

There are two types of mycobacterial resistance; acquired resistance (i.e., environmental) or by infection with a drug-resistant strain (i.e., primary resistance) [126].

Resistance is defined as an infection of a naïve patient with an already resistant tuberculosis strain 22

 Primary resistance is defined as an infection of a naive (i.e. previously untreated)

patient with an already resistant tuberculosis strain,

 Acquired resistance develops in patients originally infected with a drug-suscepti-

ble strain that during the treatment becomes resistant to multiple medications.

Therapeutic failures are primarily due to inadequate, inappropriate or irregular

treatment, non-adherence to treatment protocols and prescribed therapy or non-

compliance

These intrinsic resistance mechanisms of MTB are thought to be the crucial link between a therapeutic cure and persistent infection [119, 127]. Because virtually all classes of antibiotics require metabolically active bacteria for their primary mode of action, the non-replicating state of MTB within the host is thought to render MTB resistant to otherwise bactericidal antibiotics. This unique nature of the disease, specificity of location, and development of closed granulomas that contain persister bacilli [128, 129] increases the need for an antimicrobial treatment that provides continuous intracellular drug concentrations within the phagocyte and granuloma to be effective.

Intrinsic resistance mechanisms

The cellular wall of mycobacteria is an effective barrier against penetration of antibiotics. The cellular wall is extremely lipid-rich, multi-layered and has varied hydrophobicity. The two hydrophilic layers of Mycobacterium’s cellular wall consist of the arabinogalactan and peptidoglycan layer which both are essential in the selection and transport of hydrophobic molecules [130, 131]. Also, the waxy outer mycolic acid layer is covalently linked to the peptidoglycan and arabinogalactan layers. Together these form, 23 long chain fatty acids barrier which act as a impenetrable, non-fluidic barrier restricting the active and passive diffusion of hydrophobic and hydrophilic molecules [132, 133]

Extrinsic antibiotic resistance

Pathogenic bacteria acquire drug resistance through horizontal gene transfer through plasmids and transposons. However, in M. tuberculosis drug resistance is caused primarily by spontaneous single nucleotide polymorphism mutations in chromosomal genes producing a select strain during a suboptimal chemotherapeutic treatment [134,

135]. To date, no single pleiotropic mutation [136] has been found in Multi-drug (MDR-

TB) or Extensively drug-resistant (XDR-TB) M. tuberculosis.

M. tuberculosis mutations in the gyrA and gyrB genes are [134] phenotypically resistant but genotypically sensitive to chemotherapy [137]. This sensitivity induces spontaneous mutations within the rpoB, rrs, tlyA, katG, and embB genes and eis, inhA promoter region [134, 135, 138, 139] occurs at a rate of 10,218 mutations per cell divi- sion [140] in the presence of a single antibiotic. Like most organisms, the fitness cost associated with that of resistance is very high, but we do not observe that phenomena in

Mycobacterium [141, 142]. This high rate of genetic mutations and low fitness cost are the primary reason that a multi-drug therapy is recommended in treating tuberculosis.

Internalization

Many intracellular pathogens gain initial entry through the host mucosal surfaces, especially those of the respiratory tracts. Transmission can be primarily a result of person-to-person contact and or aerosolization of droplets containing bacteria [143], through broken skin in an aquatic environment [83] or contaminated food products [88, 24

89, 144]. Mycobacterium uses both complement and non-complement receptors to increase uptake by phagocytic cells [145, 146], where it can persist by inhibiting phagosome-lysosome maturation [147-149]. The cellular wall of Mycobacterium consists of highly lipid-rich components also perform an active role in the inhibition of endosomal maturation, and acidification of the phagolysosome.

Environmental niche and host response

Phagocytosis of M. tuberculosis by phagocytic-macrophages undergoes a series of membrane invagination, and fusion events, resulting in the formation of the phagosome.

Internalization can occur through direct surface interaction or through surface attachment through acquisition of serum opsonions to the bacterial surface. The specific route by which phagocytic entry occurs has a large impact on the survivability of M. tuberculosis once internalization occurs. The two primary routes of internalization are zipper phagocytosis [150] and tripper phagocytosis[151]. The routes by which macrophages are activate occur at varying degrees based on the specific receptor, such as mannose, scavenger, Fcγ receptors and complement. Opsonized M. tuberculosis has been shown to activate the alternative pathway of complement, resulting in opsonization with C3b molecule [152].

Complement mediated

Unlike other intracellular bacteria, mycobacteria, actively recruited complement molecules such as, C2a to form a C3 convertase and generate C3b, as well as the dendritic cell-specific intercellular adhesion molecule “(ICAM)-3-grabbing nonintegrin (DC-

SIGN)” [153, 154]. It has been suggested that internalization via the CR3 receptors pre- vents the activation of the macrophages [155]. CR3 mediated internalization requires the 25 presence of host plasma membrane cholesterol [156] Mycobacterium sequester the cholesterol to prevent the rearrangement of the membrane-cytoskeleton network to prevent fusion of the bacteria-containing vesicle with lysosomes [156, 157].

The active recruitment and sequestering of the host cytoskeleton-associated molecule coronin 1(P57) [158, 159] by mycobacteria provides another means by which inhibition of acidification of the lysosome occurs. This molecule links the cytoskeleton with the plasma membrane in leukocytes, and is equally distributed throughout non-infected macrophage. In macrophages infected with live bacilli, the mycobacteria phagolysosomes have been found to contain high levels of coronin 1 in contrast to dead bacteria found ex- clusively in lysosomes [160].

Interferon (IFN)-inducible GTPases proteins are involved in the regulation of the phagolysosome maturation by mycobacterium is IRGM1 (formerly called LRG-47)

[161]. This small GTPase protein provides pathogen specific protection [162] against intracellular infections. They primary function is in cell-autonomous resistance[163], there exact mechanism is dependent on the specific structure or organelles that the proteins interaction occurred [161, 163]. IRGM1 promotes the maturation and acidification of mycobacteria phagolysosomes [164] and regulation of cellular autophagy [165]. Autophagy can eliminate intracellular Mycobacterium in a process akin to the sequestration and degradation of large particles and dysfunctional intracellular organelles [166]. If M. tuberculosis phagosome is not appropriately acidified the cell will resort to autophagy in an at- tempt to induce an effective host response against mycobacteria [167].

Receptor mediated cellular components

Mannosylated lipoarabinomannan, lipoarabinomannan, trehalose dimycolate, and acyltrehalose containing glycolipids have all been shown to interact with several different receptors of the immune system [168-170]. Lipoarabinomannan has been associated with

Toll-like receptor signaling, Mannosylated lipoarabinomannan C-type lectins and mannose receptors of macrophages [149, 168, 169]. The microbial tyrosine phosphatase PtpA

(Rv2234) inhibits the acidification through modulation of binding of the V-ATPase with

Mycobacterium containing vacuoles [145, 168, 171-173].

Granuloma formation

During the early stage of infection, Mycobacterium replicate within macrophages and dendritic cells. While most bacteria prefer to remain within the protective confines of a macrophage Mycobacterium does not. The nature of the infectious process by which mycobacteria are spreads from host to host relies on the presence of live bacilli found within lung tissue, are vital for continual infection through aerosolization.

Alveolar infected macrophages internalize the bacilli and transport them across the lung epithelium. When an infected alveolar macrophage is recognized by CD4+ lymphocytes activation and recruitment occurs. The activated T-cells and macrophages responds by secretion of interferon-γ (INF-γ) and tumor necrosis factor-α (TNF-α) [174, 175]. This initial signaling event recruits mononuclear cells from the surrounding tissue to form the first start of the granuloma. As the localized chemokine gradient is upregulated the signaling process results in recruitment of multiple immune cell phenotypes in different states of activation [176]. This cellular recruitment is accompanied (Figure 1-4) by the 27 differentiation and activation of multinucleated giant cells, foamy macrophages, and epi- thelioid cells [177].

Figure 2-4 Granuloma comprised of aggregate immune cells [177].

The initial stages of the granuloma formation are highly vascularized, and the recruitment has little effect on Mycobacterium. As Mycobacterium replicates inflammation develops, and loss of vascularization occurs leading to necrosis of tissue with the accumulation of a caseum granuloma core[178]. It is during this stage that most mycobacteria are in the persistent state of which some remain intracellularly or within the caseum core

(Figure 1-4). Eventually, the granuloma collapses and aerosolized infectious Mycobacte- rium are expelled into the environment through coughing [179].

Treatments

The primary drugs used in the treatment of mycobacterial infections are unique between species. Because the focus of this research is on M. tuberculosis, the primary focus of antituberculous will consist of rifampicin (RIF), isoniazid (INH), pyrazinamide

(PZA) and ethambutol (EMB).

Isoniazid

Isoniazid, pyridine-4-carbohydrazide was introduced in 1952 under the name

Rimifon SV (Table 1-4) [180] [181]. It was deemed a miracle a cure for “consumption,” but the incidence of resistance began within the first six months of use [182] [183]. INH requires activation by the mycobacterial enzyme KatG. These enzymes are responsible for the generation of reactive oxygen species that are inhibitors of lipid and nucleic acid biosynthetic enzymes [184, 185]. Intrinsic resistance occurs primarily from mutations within a katG gene which encodes for the mycobacterial catalase-peroxidase enzyme that is responsible for drug activation.[186].

Table 2-4 Recommended dosage of first-line antituberculous drugs

The recommended dosage of antituberculous first-line medicines. Recommended doses of first-line antituberculotics for adults. The mechanisms of the action of a range of antimycobacterial agents are briefly summarized within the context. Drug Daily administration Administration three times per week Standard dose Maximal dose Standard dose Daily maximal dose (mg) (mg) (mg/kg) (mg/kg) Isoniazid (INH) 5 300 10 900 29

Table 2-4 continued Rifampicin 10 600 10 600 (RIF) Pyrazinamide 25 - 35 - (PZA) Ethambutol 15 - 30 - (EMB)

Rifampicin

Rifampicin, 3-(4-methyl-1-piperazinyl)-iminomethyl derivative of rifamycin SV

(Table 1-4). It was first introduced as a TB therapy in 1972. Its effective antimicrobial action, it is considered, together with isoniazid, to be the foundation of the “intensive phase”treatment regimen for TB. Rifampicin targets Mycobacterium b-subunit of RNA polymerase, where it binds and inhibits the elongation of messenger RNA [187]. Rifam- picin is active against actively growing and latent bacilli [188-190].

Pyrazinamide

Pyrazinamide, pyrazine-2-carboxamide was chemically synthesized in 1936 and not discovered as a (Figure 1-4) treatment towards tuberculosis until 1952. In its earlier use, it was considered too toxic, and the required dose was reduced to overcome this. Ad- dition of pyrazinamide to use of rifampicin and isoniazid reduced the treatment times from 9 to 6 months. Pyrazinamide is a prodrug which requires metabolic activation through conversion of its nicotinamide and its active form, pyrazinoic acid, by the enzyme pyrazinamidase/nicotinamidase (PZase) [191]. The inactive form, PZA enters My- cobacterium through passive diffusion where it is converted into the active state whereby targeting and disrupting the bacterial membrane energetics and inhibiting membrane 30 transport. In areas of high acid conditions protonated pyrazinioc acid accumulates inside the cell [131].

Ethambutol

Ethambutol, 2,2′-(1,2-ethanediyldiimino)bis-1-butanol, (Figure 1-4) was introduced in 1966 as an alternative to PZA. EMB’s activity against TB was not directly known until most recently. Together with isoniazid, rifampicin, and pyrazinamide, make up the first-line drugs currently use in the treatment of tuberculosis The use of the bacteriostatic antitubercular ethambutol (EMB), as part of the antitubercular regime was introduced in 1960 as an alternative to PZA [192]. Resistance occurs as a direct result of its overuse and poor patient compliance [193]. EMB inhibits arabinosyl transferases, en- coded on the embCAB operon, thereby inhibiting biosynthesis of the cellular wall mechanisms of arabinogalactan and lipoarabinomannan [194].

Vaccine

Bacille Calmette-Guerin (BCG) vaccine is currently the only available vaccine against MTB. Many foreign-born individuals have been BCG-vaccinated and have been administered to over three billion people without serious complications. In countries where tuberculosis or leprosy are endemic a single dose is administered to children shortly after birth [195, 196] The vaccine has been shown to provide up to 80% protection to TB meningitis[197]. Although it displays some advantages (i.e., administration, conferred immunity, and low cost), recent analysis have revealed that genetic modifications have selected for a subpopulation of resistant bacteria as a direct result of the vaccine. Additionally, there can be a gradual loss of T-memory cell population during in the 31 course of time [198]. Regrettably, BCG vaccination has had limited impact on the global burden of MTB.

References

1. Singh, J., et al., Advances in nanotechnology-based carrier systems for targeted delivery of bioactive drug molecules with special emphasis on immunotherapy in drug resistant tuberculosis - a critical review. Drug Deliv, 2016. 23(5): p. 1676- 98.

2. Wang, R., P.S. Billone, and W.M. Mullett, Nanomedicine in Action: An Overview of Cancer Nanomedicine on the Market and in Clinical Trials. Journal of Nanomaterials, 2013. 2013: p. 12.

3. Liu, S., et al., Gene and doxorubicin co-delivery system for targeting therapy of glioma. Biomaterials, 2012. 33(19): p. 4907-16.

4. Shah, M., et al., Solid lipid nanoparticles of a water soluble drug, ciprofloxacin hydrochloride. Indian Journal of Pharmaceutical Sciences, 2012. 74: p. 434.

5. Mehnert, W. and K. Mader, Solid lipid nanoparticles: production, characterization and applications. Adv Drug Deliv Rev, 2001. 47(2-3): p. 165- 96.

6. Jaiswal, M., R. Dudhe, and P.K. Sharma, Nanoemulsion: an advanced mode of drug delivery system. 3 Biotech, 2015. 5(2): p. 123-127.

7. Ahmed, K., et al., Nanoemulsion- and emulsion-based delivery systems for curcumin: Encapsulation and release properties. Food Chemistry, 2012. 132(2): p. 799-807.

8. Sun, Y.J., et al., [Preparation and characterization of nanoemulsion]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2005. 21(1): p. 69-71, 75.

9. Yilmaz, E. and H.H. Borchert, Design of a phytosphingosine-containing, positively-charged nanoemulsion as a colloidal carrier system for dermal application of ceramides. Eur J Pharm Biopharm, 2005. 60(1): p. 91-8.

10. Sadurni, N., et al., Studies on the formation of O/W nano-emulsions, by low- energy emulsification methods, suitable for pharmaceutical applications. Eur J Pharm Sci, 2005. 26(5): p. 438-45.

11. Kim, C.-K., Y.-J. Cho, and Z.-G. Gao, Preparation and evaluation of biphenyl dimethyl dicarboxylate microemulsions for oral delivery. Journal of Controlled Release, 2001. 70(1): p. 149-155. 32

12. Yukuyama, M.N., et al., Challenges and Future Prospects of Nanoemulsion as a Drug Delivery System. Curr Pharm Des, 2017. 23(3): p. 495-508.

13. Fonte, P., et al., Corrigendum to “Polymer-based nanoparticles for oral insulin delivery: Revisited approaches”,[Biotechnol Adv. 2015 Nov 1;33 (6 Pt 3):1342– 54]. Biotechnology Advances, 2016. 34(1): p. 64.

14. Danhier, F., et al., PLGA-based nanoparticles: An overview of biomedical applications. Journal of Controlled Release, 2012. 161(2): p. 505-522.

15. Petersen, L.K., A.V. Chavez-Santoscoy, and B. Narasimhan, Combinatorial Synthesis of and high-throughput protein release from polymer film and nanoparticle libraries. J Vis Exp, 2012(67).

16. Ulery, B.D., et al., Design of a Protective Single-Dose Intranasal Nanoparticle- Based Vaccine Platform for Respiratory Infectious Diseases. PLoS ONE, 2011. 6(3): p. e17642.

17. Carrillo-Conde, B., et al., Encapsulation into amphiphilic polyanhydride microparticles stabilizes Yersinia pestis antigens. Acta Biomaterialia, 2010. 6(8): p. 3110-3119.

18. Petersen, L.K., C.K. Sackett, and B. Narasimhan, Novel, High Throughput Method to Study in Vitro Protein Release from Polymer Nanospheres. Journal of Combinatorial Chemistry, 2010. 12(1): p. 51-56.

19. Kipper, M.J., et al., Design of an injectable system based on bioerodible polyanhydride microspheres for sustained drug delivery. Biomaterials, 2002. 23(22): p. 4405-12.

20. Dang, W., et al., Effects of GLIADEL® wafer initial molecular weight on the erosion of wafer and release of BCNU. Journal of Controlled Release, 1996. 42(1): p. 83-92.

21. Torres, M.P., et al., Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. Journal of Biomedical Materials Research Part A, 2006. 76A(1): p. 102-110.

22. von Burkersroda, F., L. Schedl, and A. Gopferich, Why degradable polymers undergo surface erosion or bulk erosion. Biomaterials, 2002. 23(21): p. 4221-31.

23. Ottesen, E.A., et al., The Global Programme to Eliminate Lymphatic Filariasis: Health Impact after 8 Years. PLoS Neglected Tropical Diseases, 2008. 2(10): p. e317. 33

24. Ramaiah, K.D. and E.A. Ottesen, Progress and Impact of 13 Years of the Global Programme to Eliminate Lymphatic Filariasis on Reducing the Burden of Filarial Disease. PLoS Neglected Tropical Diseases, 2014. 8(11): p. e3319.

25. Coffeng, L.E., et al., African Programme for Onchocerciasis Control 1995–2015: Model-Estimated Health Impact and Cost. PLoS Neglected Tropical Diseases, 2013. 7(1): p. e2032.

26. Grote, A., et al., Defining Brugia malayi and Wolbachia symbiosis by stage- specific dual RNA-seq. PLoS Negl Trop Dis, 2017. 11(3): p. e0005357.

27. Disease, G.B.D., I. Injury, and C. Prevalence, Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet, 2017. 390(10100): p. 1211-1259.

28. Organization, W.H. Global tuberculosis report 2017. 2017 [cited 2017 December 01]; Available from: http://www.who.int/tb/publications/global_report/en/.

29. O'Neill, M., et al., Potential Role for Flubendazole in Limiting Filariasis Transmission: Observations of Microfilarial Sensitivity. Am J Trop Med Hyg, 2017.

30. Paily, K.P., S.L. Hoti, and P.K. Das, A review of the complexity of biology of lymphatic filarial parasites. Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology, 2009. 33(1-2): p. 3-12.

31. Control, C.f.D. Parasites - Lymphatic Filariasis. 2010 June 14, 2013 [cited 2017 December 11]; Available from: https://www.cdc.gov/parasites/lymphaticfilariasis/biology.html.

32. Buck, A.H., et al., Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity. Nat Commun, 2014. 5: p. 5488.

33. McSorley, H.J., J.P. Hewitson, and R.M. Maizels, Immunomodulation by helminth parasites: defining mechanisms and mediators. Int J Parasitol, 2013. 43(3-4): p. 301-10.

34. Greene, B.M., H.R. Taylor, and M. Aikawa, Cellular killing of microfilariae of Onchocerca volvulus: eosinophil and neutrophil-mediated immune serum- dependent destruction. J Immunol, 1981. 127(4): p. 1611-8.

35. McCoy, C.J., et al., Human Leukocytes Kill Brugia malayi Microfilariae Independently of DNA-Based Extracellular Trap Release. PLoS Negl Trop Dis, 2017. 11(1): p. e0005279. 34

36. Schroeder, J.H., et al., Live Brugia malayi microfilariae inhibit transendothelial migration of neutrophils and monocytes. PLoS Negl Trop Dis, 2012. 6(11): p. e1914.

37. Bandi, C., et al., Phylogeny of Wolbachia in filarial nematodes. Proceedings of the Royal Society B: Biological Sciences, 1998. 265(1413): p. 2407-2413.

38. Bowman, D.D., Introduction to the Alpha-proteobacteria: Wolbachia and Bartonella, Rickettsia, Brucella, Ehrlichia, and Anaplasma. Topics in Companion Animal Medicine. 26(4): p. 173-177.

39. Bouchery, T., et al., The symbiotic role of Wolbachia in Onchocercidae and its impact on filariasis. Clinical Microbiology and Infection. 19(2): p. 131-140.

40. Bandi, C., et al., Phylogeny of Wolbachia in filarial nematodes. Proc Biol Sci, 1998. 265(1413): p. 2407-13.

41. Hoerauf, A., et al., Filariasis in Africa--treatment challenges and prospects. Clin Microbiol Infect, 2011. 17(7): p. 977-85.

42. Maizels, R.M. and D.A. Denham, Diethylcarbamazine (DEC): immunopharmacological interactions of an anti-filarial drug. Parasitology, 1992. 105 Suppl: p. S49-60.

43. Binnebose, A.M., et al., Polyanhydride Nanoparticle Delivery Platform Dramatically Enhances Killing of Filarial Worms. PLoS Negl Trop Dis, 2015. 9(10): p. e0004173.

44. Molyneux, D.H., et al., Mass drug treatment for lymphatic filariasis and onchocerciasis. Trends Parasitol, 2003. 19(11): p. 516-22.

45. Supali, T., et al., Doxycycline treatment of Brugia malayi-infected persons reduces microfilaremia and adverse reactions after diethylcarbamazine and albendazole treatment. Clin Infect Dis, 2008. 46(9): p. 1385-93.

46. Talbot, J.T., et al., Predictors of compliance in mass drug administration for the treatment and prevention of lymphatic filariasis in Leogane, Haiti. Am J Trop Med Hyg, 2008. 78(2): p. 283-8.

47. Al-Abd, N.M., et al., Antifilarial and Antibiotic Activities of Methanolic Extracts of Melaleuca cajuputi Flowers. Korean J Parasitol, 2016. 54(3): p. 273-80.

48. Vatta, A.F., et al., Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro. Vet Parasitol, 2014. 206(1-2): p. 38-42.

49. Poolman, E.M. and A.P. Galvani, Modeling targeted ivermectin treatment for controlling river blindness. Am J Trop Med Hyg, 2006. 75(5): p. 921-7.

50. Yamey, G., Global alliance launches plan to eliminate lymphatic filariasis. BMJ, 2000. 320(7230): p. 269.

51. Sungpradit, S., T. Chatsuwan, and S. Nuchprayoon, Susceptibility of Wolbachia, an endosymbiont of Brugia malayi microfilariae, to doxycycline determined by quantitative PCR assay. Southeast Asian J Trop Med Public Health, 2012. 43(4): p. 841-50.

52. Turner, J.D., et al., Macrofilaricidal activity after doxycycline only treatment of Onchocerca volvulus in an area of Loa loa co-endemicity: a randomized controlled trial. PLoS Negl Trop Dis, 2010. 4(4): p. e660.

53. Taylor, M.J., et al., Macrofilaricidal activity after doxycycline treatment of Wuchereria bancrofti: a double-blind, randomised placebo-controlled trial. Lancet, 2005. 365(9477): p. 2116-21.

54. Hoerauf, A., et al., Endosymbiotic bacteria in worms as targets for a novel chemotherapy in filariasis. Lancet, 2000. 355(9211): p. 1242-3.

55. Townson, S., et al., Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro. Filaria J, 2006. 5: p. 4.

56. Hoerauf, A., et al., Efficacy of 5-week doxycycline treatment on adult Onchocerca volvulus. Parasitol Res, 2009. 104(2): p. 437-47.

57. Specht, S., et al., Efficacy of 2- and 4-week rifampicin treatment on the Wolbachia of Onchocerca volvulus. Parasitol Res, 2008. 103(6): p. 1303-9.

58. Tamarozzi, F., et al., Long term impact of large scale community-directed delivery of doxycycline for the treatment of onchocerciasis. Parasit Vectors, 2012. 5: p. 53.

59. Campbell, W.C., Ivermectin as an Antiparasitic Agent for Use in Humans. Annual Review of Microbiology, 1991. 45(1): p. 445-474.

60. Ballesteros, C., et al., The Effects of Ivermectin on Brugia malayi Females In Vitro: A Transcriptomic Approach. PLoS Neglected Tropical Diseases, 2016. 10(8): p. e0004929. 36

61. Abongwa, M., R.J. Martin, and A.P. Robertson, A BRIEF REVIEW ON THE MODE OF ACTION OF ANTINEMATODAL DRUGS. Acta veterinaria, 2017. 67(2): p. 137-152.

62. Cully, D.F., et al., Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans. Nature, 1994. 371: p. 707.

63. Carithers, D.S., Examining the role of macrolides and host immunity in combatting filarial parasites. Parasites & Vectors, 2017. 10: p. 182.

64. Wolstenholme, A.J. and A.T. Rogers, Glutamate-gated chloride channels and the mode of action of the avermectin/milbemycin anthelmintics. Parasitology, 2005. 131(S1): p. S85-S95.

65. Fatma, N., K.B. Mathur, and R.K. Chatterjee, Chemotherapy of experimental filariasis: Enhancement of activity profile of ivermectin with immunomodulators. Acta Tropica, 1994. 57(1): p. 55-67.

66. Griffin, M.O., et al., Tetracyclines: a pleitropic family of compounds with promising therapeutic properties. Review of the literature. American Journal of Physiology - Cell Physiology, 2010. 299(3): p. C539-C548.

67. Aljayyoussi, G., et al., Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis. Scientific Reports, 2017. 7: p. 210.

68. Debrah, A.Y., et al., Doxycycline Leads to Sterility and Enhanced Killing of Female Onchocerca volvulus Worms in an Area With Persistent Microfilaridermia After Repeated Ivermectin Treatment: A Randomized, Placebo- Controlled, Double-Blind Trial. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America, 2015. 61(4): p. 517- 526.

69. Taylor, M.J., A. Hoerauf, and M. Bockarie, Lymphatic filariasis and onchocerciasis. The Lancet. 376(9747): p. 1175-1185.

70. Chirgwin, S.R., et al., Removal of Wolbachia from Brugia pahangi Is Closely Linked to Worm Death and Fecundity but Does Not Result in Altered Lymphatic Lesion Formation in Mongolian Gerbils (Meriones unguiculatus). Infection and Immunity, 2003. 71(12): p. 6986-6994.

71. Taylor, M.J. and A. Hoerauf, A new approach to the treatment of filariasis. Curr Opin Infect Dis, 2001. 14(6): p. 727-31.

72. Lacey, E., Mode of action of benzimidazoles. Parasitology Today, 1990. 6(4): p. 112-115.

73. Borgsteede, F.H.M., The activity of albendazole against adult and larval gastrointestinal nematodes in naturally infected calves in the Netherlands. Veterinary Quarterly, 1979. 1(4): p. 181-188.

74. Aguayo-Ortiz, R., et al., Towards the identification of the binding site of benzimidazoles to β-tubulin of Trichinella spiralis: insights from computational and experimental data. Journal of molecular graphics & modelling, 2013. 41: p. 12-19.

75. Kwarteng, A., S.T. Ahuno, and F.O. Akoto, Killing filarial nematode parasites: role of treatment options and host immune response. Infectious Diseases of Poverty, 2016. 5: p. 86.

76. Thomsen, E.K., et al., Efficacy, Safety, and Pharmacokinetics of Coadministered Diethylcarbamazine, Albendazole, and Ivermectin for Treatment of Bancroftian Filariasis. Clinical Infectious Diseases, 2016. 62(3): p. 334-341.

77. Meyrowitsch, D.W. and P.E. Simonsen, Short communication: Efficacy of DEC against Ascaris and hookworm infections in schoolchildren. Tropical Medicine & International Health, 2001. 6(9): p. 739-742.

78. Peixoto, C.A. and B.S. Silva, Anti-inflammatory effects of diethylcarbamazine: A review. European Journal of Pharmacology, 2014. 734: p. 35-41.

79. Li, Q., et al., Cardioprotection afforded by inducible nitric oxide synthase gene therapy is mediated by cyclooxygenase-2 via a nuclear factor-κB-dependent pathway. Circulation, 2007. 116(14): p. 1577-1584.

80. Patel, S.S., et al., Necrotizing Soft Tissue Infection Occurring after Exposure to Mycobacterium marinum. Case Rep Infect Dis, 2014. 2014: p. 702613.

81. Griffith, D.E., et al., An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med, 2007. 175(4): p. 367-416.

82. Rallis, E. and E. Koumantaki-Mathioudaki, Treatment of Mycobacterium marinum cutaneous infections. Expert Opin Pharmacother, 2007. 8(17): p. 2965- 78.

83. Lewis, F.M., B.J. Marsh, and C.F. von Reyn, Fish tank exposure and cutaneous infections due to Mycobacterium marinum: tuberculin skin testing, treatment, and prevention. Clin Infect Dis, 2003. 37(3): p. 390-7.

84. Stevenson, K., Genetic diversity of Mycobacterium avium subspecies paratuberculosis and the influence of strain type on infection and pathogenesis: a review. Vet Res, 2015. 46: p. 64.

85. Bernstein, C.N., et al., Population-Based Case Control Study of Seroprevalence of Mycobacterium paratuberculosis in Patients with Crohn's Disease and Ulcerative Colitis. Journal of Clinical Microbiology, 2004. 42(3): p. 1129-1135.

86. Collins, M.T., et al., Results of Multiple Diagnostic Tests for Mycobacterium avium subsp. paratuberculosis in Patients with Inflammatory Bowel Disease and in Controls. Journal of Clinical Microbiology, 2000. 38(12): p. 4373-4381.

87. Lee, A., et al., Association of Mycobacterium avium subspecies paratuberculosis with Crohn Disease in pediatric patients. J Pediatr Gastroenterol Nutr, 2011. 52(2): p. 170-4.

88. Pierce, E.S., Could Mycobacterium avium subspecies paratuberculosis cause Crohn’s disease, ulcerative colitis…and colorectal cancer? Infectious Agents and Cancer, 2018. 13: p. 1.

89. Timms, V.J., et al., The Association of Mycobacterium avium subsp. paratuberculosis with Inflammatory Bowel Disease. PLoS ONE, 2016. 11(2): p. e0148731.

90. Whittington, R.J., et al., Case definition terminology for paratuberculosis (Johne's disease). BMC Vet Res, 2017. 13(1): p. 328.

91. Bates, A., et al., The effect of sub-clinical infection with Mycobacterium avium subsp. paratuberculosis on milk production in a New Zealand dairy herd. BMC Vet Res, 2018. 14(1): p. 93.

92. Whittington, R.J., et al., Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: IS900 restriction fragment length polymorphism and IS1311 polymorphism analyses of isolates from animals and a human in Australia. J Clin Microbiol, 2000. 38(9): p. 3240-8.

93. United States Department of Agriculture. 2017 Aug 18, 2017 03/18/2018]; Johne's Disease]. Available from: https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/nvap/NVAP-Reference- Guide/Control-and-Eradication/Johnes-Disease.

94. Patel, A. and N. Shah, Mycobacterium avium subsp paratuberculosis--incidences in milk and milk products, their isolation, enumeration, characterization, and role in human health. J Microbiol Immunol Infect, 2011. 44(6): p. 473-9.

95. Pribylova, R., et al., Mycobacterium avium subsp. Paratuberculosis and the expression of selected virulence and pathogenesis genes in response to 6 degrees C, 65 degrees c and ph 2.0. Braz J Microbiol, 2011. 42(2): p. 807-17.

96. Pickup, R.W., et al., Mycobacterium avium subsp. paratuberculosis in lake catchments, in river water abstracted for domestic use, and in effluent from domestic sewage treatment works: diverse opportunities for environmental cycling and human exposure. Appl Environ Microbiol, 2006. 72(6): p. 4067-77.

97. Slater, N., et al., Impact of the shedding level on transmission of persistent infections in Mycobacterium avium subspecies paratuberculosis (MAP). Vet Res, 2016. 47: p. 38.

98. Pribylova, R., et al., Effect of short- and long-term antibiotic exposure on the viability of Mycobacterium avium subsp. paratuberculosis as measured by propidium monoazide F57 real time quantitative PCR and culture. Vet J, 2012. 194(3): p. 354-60.

99. Krishnan, M.Y., E.J.B. Manning, and M.T. Collins, Comparison of three methods for susceptibility testing of Mycobacterium avium subsp. paratuberculosis to 11 antimicrobial drugs. Journal of Antimicrobial Chemotherapy, 2009. 64(2): p. 310-316.

100. Hermon-Taylor, J., Treatment with drugs active against Mycobacterium avium subspecies paratuberculosis can heal Crohn's disease: more evidence for a neglected Public Health tragedy. Digestive and Liver Disease. 34(1): p. 9-12.

101. Piersimoni, C., et al., Evaluation of a New Method for Rapid Drug Susceptibility Testing of Mycobacterium avium Complex Isolates by Using the Mycobacteria Growth Indicator Tube. Journal of Clinical Microbiology, 1998. 36(1): p. 64-67.

102. Klopper, M., et al., Emergence and Spread of Extensively and Totally Drug- Resistant Tuberculosis, South Africa. Emerging Infectious Diseases, 2013. 19(3): p. 449-455.

103. Ducati, R.G., et al., The resumption of consumption -- a review on tuberculosis. Mem Inst Oswaldo Cruz, 2006. 101(7): p. 697-714.

104. McGrath, E.E., et al., Nontuberculous mycobacteria and the lung: from suspicion to treatment. Lung, 2010. 188(4): p. 269-82.

105. Aderem, A. and D.M. Underhill, Mechanisms of phagocytosis in macrophages. Annu Rev Immunol, 1999. 17: p. 593-623.

106. Jimenez-Fuentes, M.A., et al., Treatment of Latent Tuberculosis Infection in a Tuberculosis Clinic. Arch Bronconeumol, 2018.

107. Tang, P. and J. Johnston, Treatment of Latent Tuberculosis Infection. Curr Treat Options Infect Dis, 2017. 9(4): p. 371-379.

108. Anguru, M.R., et al., Novel drug targets for Mycobacterium tuberculosis: 2- heterostyrylbenzimidazoles as inhibitors of cell wall protein synthesis. Chem Cent J, 2017. 11(1): p. 68.

109. Tomioka, H., New approaches to tuberculosis--novel drugs based on drug targets related to toll-like receptors in macrophages. Curr Pharm Des, 2014. 20(27): p. 4404-17.

110. Mortellaro, A., L. Robinson, and P. Ricciardi-Castagnoli, Spotlight on Mycobacteria and dendritic cells: will novel targets to fight tuberculosis emerge? EMBO Mol Med, 2009. 1(1): p. 19-29.

111. Saravanan, M., et al., Review on emergence of drug-resistant tuberculosis (MDR & XDR-TB) and its molecular diagnosis in Ethiopia. Microb Pathog, 2018. 117: p. 237-242.

112. Berrada, Z.L., et al., Rifabutin and rifampin resistance levels and associated rpoB mutations in clinical isolates of Mycobacterium tuberculosis complex. Diagn Microbiol Infect Dis, 2016. 85(2): p. 177-81.

113. Jamieson, F., et al., Profiling of rpoB Mutations and MICs for Rifampin and Rifabutin in Mycobacterium tuberculosis. Journal of Clinical Microbiology, 2014. 52(6): p. 2157.

114. Namouchi, A., et al., Phenotypic and genomic comparison of Mycobacterium aurum and surrogate model species to Mycobacterium tuberculosis: implications for drug discovery. BMC Genomics, 2017. 18: p. 530.

115. Dharra, R., et al., Rational design of drug-like compounds targeting Mycobacterium marinum MelF protein. PLoS ONE, 2017. 12(9): p. e0183060.

116. Forrellad, M.A., et al., Virulence factors of the Mycobacterium tuberculosis complex. Virulence, 2013. 4(1): p. 3-66.

117. Hoagland, D.T., et al., New agents for the treatment of drug-resistant Mycobacterium tuberculosis. Advanced Drug Delivery Reviews, 2016. 102: p. 55-72.

118. Izadi, N., et al., Co-infection of long-standing extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) and non-tuberculosis mycobacteria: A case report. Respiratory Medicine Case Reports, 2015. 15: p. 12-13.

119. Prat, C. and A. Lacoma, Bacteria in the respiratory tract-how to treat? Or do not treat? Int J Infect Dis, 2016. 51: p. 113-122.

120. Connolly, L.E., P.H. Edelstein, and L. Ramakrishnan, Why Is Long-Term Therapy Required to Cure Tuberculosis? PLoS Medicine, 2007. 4(3): p. e120.

121. Vandiviere, H.M., et al., The treated pulmonary lesion and its tubercle bacillus. II. The death and resurrection. Am J Med Sci, 1956. 232(1): p. 30-7; passim.

122. Ahmad, Z., et al., Mycobacterium tuberculosis Effectiveness of tuberculosis chemotherapy correlates with resistance to Mycobacterium tuberculosis infection in animal models. Journal of Antimicrobial Chemotherapy, 2011. 66(7): p. 1560- 1566.

123. Smith, T., K.A. Wolff, and L. Nguyen, Molecular Biology of Drug Resistance in Mycobacterium tuberculosis. Current topics in microbiology and immunology, 2013. 374: p. 53-80.

124. Jing, W., et al., Rifabutin Resistance Associated with Double Mutations in rpoB Gene in Mycobacterium tuberculosis Isolates. Front Microbiol, 2017. 8: p. 1768.

125. Yinnon, A.M., et al., Emergence of drug-resistant tuberculosis in Jerusalem: ten- year retrospective review. Isr J Med Sci, 1997. 33(11): p. 728-33.

126. Shah, N.S., et al., Worldwide Emergence of Extensively Drug-resistant Tuberculosis. Emerging Infectious Disease journal, 2007. 13(3): p. 380.

127. Nguyen, L., Antibiotic resistance mechanisms in M. tuberculosis: an update. Archives of toxicology, 2016. 90(7): p. 1585-1604.

128. Agrawal, N., et al., Dissecting host factors that regulate the early stages of tuberculosis infection. Tuberculosis, 2016. 100: p. 102-113.

129. O'Garra, A., et al., The Immune Response in Tuberculosis. Annual Review of Immunology, 2013. 31(1): p. 475-527.

130. Brennan, P.J. and H. Nikaido, The Envelope of Mycobacteria. Annual Review of Biochemistry, 1995. 64(1): p. 29-63.

131. Smith, T., K.A. Wolff, and L. Nguyen, Molecular biology of drug resistance in Mycobacterium tuberculosis. Curr Top Microbiol Immunol, 2013. 374: p. 53-80.

132. Marrakchi, H., M.A. Laneelle, and M. Daffe, Mycolic acids: structures, biosynthesis, and beyond. Chem Biol, 2014. 21(1): p. 67-85.

133. Tsai, Y.T., et al., Role of long-chain acyl-CoAs in the regulation of mycolic acid biosynthesis in mycobacteria. Open Biol, 2017. 7(7).

134. Oudghiri, A., et al., Molecular characterization of mutations associated with resistance to second-line tuberculosis drug among multidrug-resistant tuberculosis patients from high prevalence tuberculosis city in Morocco. BMC Infect Dis, 2018. 18(1): p. 98.

135. Mekonnen, D., et al., Multidrug-resistant and heteroresistant Mycobacterium tuberculosis and associated gene mutations in Ethiopia. Int J Infect Dis, 2015. 39: p. 34-8.

136. Almeida Da Silva, P.E. and J.C. Palomino, Molecular basis and mechanisms of drug resistance in Mycobacterium tuberculosis: classical and new drugs. Journal of Antimicrobial Chemotherapy, 2011. 66(7): p. 1417-1430.

137. Takawira, F.T., et al., Mutations in rpoB and katG genes of multidrug resistant mycobacterium tuberculosis undetectable using genotyping diagnostic methods. Pan Afr Med J, 2017. 27: p. 145.

138. Zhao, L.L., et al., Analysis of embCAB mutations associated with ethambutol resistance in multidrug-resistant mycobacterium tuberculosis isolates from China. Antimicrob Agents Chemother, 2015. 59(4): p. 2045-50.

139. Zhang, Z., et al., Ethambutol resistance as determined by broth dilution method correlates better than sequencing results with embB mutations in multidrug- resistant Mycobacterium tuberculosis isolates. J Clin Microbiol, 2014. 52(2): p. 638-41.

140. Salvatore, P.P., et al., Fitness Costs of Drug Resistance Mutations in Multidrug- Resistant Mycobacterium tuberculosis: A Household-Based Case-Control Study. J Infect Dis, 2016. 213(1): p. 149-55.

141. Salvatore, P.P., et al., Fitness Costs of Drug Resistance Mutations in Multidrug- Resistant Mycobacterium tuberculosis: A Household-Based Case-Control Study. The Journal of Infectious Diseases, 2016. 213(1): p. 149-155.

142. Gagneux, S., et al., Impact of Bacterial Genetics on the Transmission of Isoniazid-Resistant Mycobacterium tuberculosis. PLoS Pathogens, 2006. 2(6): p. e61.

143. Johnson, M.M. and J.A. Odell, Nontuberculous mycobacterial pulmonary infections. Journal of Thoracic Disease, 2014. 6(3): p. 210-220. 43

144. Kuenstner, J.T., et al., Resolution of Crohn's disease and complex regional pain syndrome following treatment of paratuberculosis. World Journal of Gastroenterology : WJG, 2015. 21(13): p. 4048-4062.

145. Souza, C.D., et al., Cell membrane receptors on bovine mononuclear phagocytes involved in phagocytosis of Mycobacterium avium subsp paratuberculosis. Am J Vet Res, 2007. 68(9): p. 975-80.

146. Woo, S.R., et al., Bovine monocytes and a macrophage cell line differ in their ability to phagocytose and support the intracellular survival of Mycobacterium avium subsp. paratuberculosis. Vet Immunol Immunopathol, 2006. 110(1-2): p. 109-20.

147. Cheville, N.F., et al., Intracellular trafficking of Mycobacterium avium ss. paratuberculosis in macrophages. Dtsch Tierarztl Wochenschr, 2001. 108(6): p. 236-43.

148. Kuehnel, M.P., et al., Characterization of the intracellular survival of Mycobacterium avium ssp. paratuberculosis: phagosomal pH and fusogenicity in J774 macrophages compared with other mycobacteria. Cell Microbiol, 2001. 3(8): p. 551-66.

149. Keown, D.A., D.A. Collings, and J.I. Keenan, Uptake and persistence of Mycobacterium avium subsp. paratuberculosis in human monocytes. Infect Immun, 2012. 80(11): p. 3768-75.

150. Tollis, S., et al., The zipper mechanism in phagocytosis: energetic requirements and variability in phagocytic cup shape. BMC Systems Biology, 2010. 4: p. 149- 149.

151. Swanson, J.A. and S.C. Baer, Phagocytosis by zippers and triggers. Trends in Cell Biology, 1995. 5(3): p. 89-93.

152. Schlesinger, L.S., et al., Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3. The Journal of Immunology, 1990. 144(7): p. 2771.

153. Chen, P., et al., A highly efficient Ziehl-Neelsen stain: Identifying de novo intracellular Mycobacterium tuberculosis and improving detection of extracellular M. tuberculosis in cerebrospinal fluid. Journal of Clinical Microbiology, 2012. 50(4): p. 1166-1170.

154. Zhang, M., et al., Active phagocytosis of Mycobacterium tuberculosis (H37Ra) by T lymphocytes (Jurkat cells). Molecular Immunology, 2015. 66(2): p. 429-438.

155. Caron, E. and A. Hall, Identification of Two Distinct Mechanisms of Phagocytosis Controlled by Different Rho GTPases. Science, 1998. 282(5394): p. 1717.

156. Gatfield, J. and J. Pieters, Essential Role for Cholesterol in Entry of Mycobacteria into Macrophages. Science, 2000. 288(5471): p. 1647.

157. Jayachandran, R., et al., Survival of Mycobacteria in Macrophages Is Mediated by Coronin 1-Dependent Activation of Calcineurin. Cell. 130(1): p. 37-50.

158. Gatfield, J., et al., Association of the leukocyte plasma membrane with the actin cytoskeleton through coiled coil-mediated trimeric coronin 1 molecules. Molecular Biology of the Cell, 2005. 16(6): p. 2786-2798.

159. Ferrari, G., et al., A Coat Protein on Phagosomes Involved in the Intracellular Survival of Mycobacteria. Cell. 97(4): p. 435-447.

160. Tailleux, L., et al., Constrained Intracellular Survival of <em>Mycobacterium tuberculosis</em> in Human Dendritic Cells. The Journal of Immunology, 2003. 170(4): p. 1939.

161. Hunn, J.P., et al., The immunity-related GTPases in mammals: a fast-evolving cell-autonomous resistance system against intracellular pathogens. Mammalian genome : official journal of the International Mammalian Genome Society, 2011. 22(1-2): p. 43-54.

162. Ritchie, J.A., et al., Host interferon-gamma inducible protein contributes to Brucella survival. Front Cell Infect Microbiol, 2012. 2: p. 55.

163. Springer, H.M., et al., Irgm1 (LRG-47), a regulator of cell-autonomous immunity, does not localize to mycobacterial or listerial phagosomes in IFN-γ-induced mouse cells(). Journal of immunology (Baltimore, Md. : 1950), 2013. 191(4): p. 1765-1774.

164. Singh, S.B., et al., Human IRGM Induces Autophagy to Eliminate Intracellular Mycobacteria. Science, 2006. 313(5792): p. 1438.

165. Deretic, V., et al., Autophagy in Immunity Against Mycobacterium tuberculosis: a Model System to Dissect Immunological Roles of Autophagy. Current topics in microbiology and immunology, 2009. 335: p. 169-188.

166. Gutierrez, M.G., et al., Autophagy Is a Defense Mechanism Inhibiting BCG and Mycobacterium tuberculosis Survival in Infected Macrophages. Cell. 119(6): p. 753-766.

167. Jo, E.-K., Autophagy as an innate defense against mycobacteria. Pathogens and Disease, 2013. 67(2): p. 108-118. 45

168. Queval, C.J., et al., Mycobacterium tuberculosis Controls Phagosomal Acidification by Targeting CISH-Mediated Signaling. Cell Rep, 2017. 20(13): p. 3188-3198.

169. Mishra, A.K., et al., Lipoarabinomannan and related glycoconjugates: structure, biogenesis and role in Mycobacterium tuberculosis physiology and host-pathogen interaction. FEMS Microbiol Rev, 2011. 35(6): p. 1126-57.

170. Via, L.E., et al., Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7. J Biol Chem, 1997. 272(20): p. 13326-31.

171. Clemens, D.L., B.Y. Lee, and M.A. Horwitz, Mycobacterium tuberculosis and Legionella pneumophila phagosomes exhibit arrested maturation despite acquisition of Rab7. Infect Immun, 2000. 68(9): p. 5154-66.

172. Vergne, I., et al., Mycobacterium tuberculosis phagosome maturation arrest: mycobacterial phosphatidylinositol analog phosphatidylinositol mannoside stimulates early endosomal fusion. Mol Biol Cell, 2004. 15(2): p. 751-60.

173. Johansen, P., et al., Relief from Zmp1-mediated arrest of phagosome maturation is associated with facilitated presentation and enhanced immunogenicity of mycobacterial antigens. Clin Vaccine Immunol, 2011. 18(6): p. 907-13.

174. Pagán, A.J. and L. Ramakrishnan, Immunity and Immunopathology in the Tuberculous Granuloma. Cold Spring Harbor Perspectives in Medicine, 2015. 5(9): p. a018499.

175. Ufimtseva, E., Mycobacterium-Host Cell Relationships in Granulomatous Lesions in a Mouse Model of Latent Tuberculous Infection. BioMed Research International, 2015. 2015: p. 948131.

176. Marino, S., et al., Macrophage Polarization Drives Granuloma Outcome during Mycobacterium tuberculosis Infection. Infection and Immunity, 2015. 83(1): p. 324-338.

177. Ehlers, S. and U.E. Schaible, The Granuloma in Tuberculosis: Dynamics of a Host–Pathogen Collusion. Frontiers in Immunology, 2012. 3: p. 411.

178. Prosser, G., et al., The bacillary and macrophage response to hypoxia in tuberculosis and the consequences for T cell antigen recognition. Microbes and Infection, 2017. 19(3): p. 177-192.

179. Cambier, C.J., S. Falkow, and L. Ramakrishnan, Host evasion and exploitation schemes of Mycobacterium tuberculosis. Cell, 2014. 159(7): p. 1497-509. 46

180. National Center for Biotechnology Information. PubChem Compound Database; CID=1046,. [cited 2018 March 21]; Available from: https://pubchem.ncbi.nlm.nih.gov/compound/1046

181. Rimbaud, P., F. Duntze, and S. Fabre, [Effects of isonicotinic acid hydrazide (rimifon) in 2 cases of pemphigus vulgaris]. Bull Soc Fr Dermatol Syphiligr, 1958. 65(3): p. 296-7.

182. Krakauer, R., [The therapy of cutaneous tuberculosis with INH (rimifon) from 1952 to 1959 at the Dermatologische Klinik Zuerich]. Dermatologica, 1960. 120: p. 323-44.

183. Torres, J.N., et al., Novel katG mutations causing isoniazid resistance in clinical M. tuberculosis isolates. Emerg Microbes Infect, 2015. 4(7): p. e42.

184. Unissa, A.N., et al., Overview on mechanisms of isoniazid action and resistance in Mycobacterium tuberculosis. Infect Genet Evol, 2016. 45: p. 474-492.

185. Timmins, G.S. and V. Deretic, Mechanisms of action of isoniazid. Mol Microbiol, 2006. 62(5): p. 1220-7.

186. Bernardes-Genisson, V., et al., Isoniazid: an update on the multiple mechanisms for a singular action. Curr Med Chem, 2013. 20(35): p. 4370-85.

187. Patel, Y.S. and S. Mehra, Synergistic Response of Rifampicin with Hydroperoxides on Mycobacterium: A Mechanistic Study. Front Microbiol, 2017. 8: p. 2075.

188. Lavania, M., et al., Enriched whole genome sequencing identified compensatory mutations in the RNA polymerase gene of rifampicin-resistant Mycobacterium leprae strains. Infect Drug Resist, 2018. 11: p. 169-175.

189. Gupta, R., et al., Isoniazid and rifampicin heteroresistant Mycobacterium tuberculosis isolated from tuberculous meningitis patients in India. Indian J Tuberc, 2018. 65(1): p. 52-56.

190. Jaleta, K.N., et al., Rifampicin-resistant Mycobacterium tuberculosis among tuberculosis-presumptive cases at University of Gondar Hospital, northwest Ethiopia. Infect Drug Resist, 2017. 10: p. 185-192.

191. Zhang, Y., et al., Mechanisms of Pyrazinamide Action and Resistance. Microbiology spectrum, 2013. 2(4): p. 1-12.

192. Park, Y.K., et al., Correlation of the phenotypic ethambutol susceptibility of Mycobacterium tuberculosis with embB gene mutations in Korea. J Med Microbiol, 2012. 61(Pt 4): p. 529-34.

193. Shi, D., et al., Characteristics of embB mutations in multidrug-resistant Mycobacterium tuberculosis isolates in Henan, China. J Antimicrob Chemother, 2011. 66(10): p. 2240-7.

194. Telenti, A., et al., The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol. Nat Med, 1997. 3(5): p. 567-70.

195. Zimmermann, P., A. Finn, and N. Curtis, Does BCG Vaccination Protect Against Non-Tuberculous Mycobacterial Infection? A Systematic Review and Meta- Analysis. The Journal of Infectious Diseases, 2018: p. jiy207-jiy207.

196. Stensballe, L.G., et al., BCG Vaccination at Birth and Rate of Hospitalization for Infection Until 15 Months of Age in Danish Children: A Randomized Clinical Multicenter Trial. Journal of the Pediatric Infectious Diseases Society, 2018: p. piy029-piy029.

197. Counoupas, C., et al., Protective efficacy of recombinant BCG over-expressing protective, stage-specific antigens of Mycobacterium tuberculosis. Vaccine. 198. Martin, C., N. Aguilo, and J. Gonzalo-Asensio, Vacunación frente a tuberculosis. Enfermedades Infecciosas y Microbiología Clínica.

CHAPTER 3 . POLYANHYDRIDE NANOPARTICLE DELIVERY PLATFORM DRAMATICALLY ENHANCES KILLING OF FILARIAL WORMS

Reprinted from PLoS Neglected Tropical Diseases 10.1371/journal.pntd.0004173, 2015

Andrea M. Binnebose1, Shannon L. Haughney2, Richard Martin3, Paula M. Imerman4, Balaji Narasimhan2*, and Bryan H. Bellaire1* 1 Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, 50011 2 Department of Chemical and Biological Engineering, Iowa State University, Ames, 50011 3Department of Biomedical Sciences, Iowa State University, Ames, 50011 4 Department of Veterinary Diagnostic Laboratory, Iowa State University, Ames, 50011

* To whom all correspondence must be addressed

E-mail: [email protected]; Phone: (515) 294-1006, [email protected] Phone: (515) 294-8019

Abstract

Filarial diseases represent a significant social and economic burden to over 120 million people worldwide and are caused by endoparasites that require the presence of symbiotic bacteria of the genus Wolbachia for fertility and viability of the host parasite.

Targeting Wolbachia for elimination is a therapeutic approach that shows promise in the treatment of onchocerciasis and lymphatic filariasis. Here we demonstrate the use of a biodegradable polyanhydride nanoparticle-based platform for the co-delivery of the antibiotic doxycycline with the antiparasitic drug, ivermectin, to reduce microfilarial

49 burden and rapidly kill adult worms. When doxycycline and ivermectin were co- delivered within polyanhydride nanoparticles, effective killing of adult female Brugia malayi filarial worms was achieved with approximately 4,000-fold reduction in the amount of drug used. Additionally the time to death of the macrofilaria was also significantly reduced (five-fold) when the anti-filarial drug cocktail was delivered within polyanhydride nanoparticles. We hypothesize that the mechanism behind this dramatically enhanced killing of the macrofilaria is the ability of the polyanhydride nanoparticles to behave as a Trojan horse and penetrate the cuticle, bypassing excretory pumps of B. malayi, and effectively deliver drug directly to both the worm and

Wolbachia at high enough microenvironmental concentrations to cause death. These provocative findings may have significant consequences for the reduction in the amount of drug and the length of treatment required for filarial infections in terms of patient compliance and reduced cost of treatment.

Author Summary

Infection with the filarial endoparasites Brugia malayi and its symbiotic bacteria

Wolbachia represent a significant burden to both humans and animals. Current treatment protocols include use of multiple drugs over a course of months to years, resulting in high costs, undesirable side effects, and poor patient compliance. By encapsulating two of these drugs, ivermectin and doxycycline, into biodegradable polyanhydride nanoparticles, we report the ability to effectively kill adult B. malayi with up to a 4,000-fold reduction

50 in the amount of drug used. These results demonstrate a promising role for the use of nanoscale drug carriers to reduce both the course of treatment and the amount of drug needed to increase affordability of lymphatic filariasis treatment and enhance patient compliance.

Introduction

Filarial parasites belonging to the family Onchocercidae remain a significant global burden, endemic in over 80 countries worldwide, particularly India and sub-

Saharan Africa [1] , and infecting up to 120 million individuals [2]. The major human diseases caused by filaria are Lymphatic Filariasis (LF), River Blindness (RB), and

Loiasis. Of the Onchocercidae, Onchocerca volvulus causes RB while LF results from the infections of Wuchereria bancrofti and Brugia malayi. Infection is initiated through arthropod transmission of larvae into the skin of a vertebrate host where they feed, mature into fertile adults and reproduce. Adult worms survive within these tissues for several years shedding thousands of microfilariae (MF) that migrate through the skin and lymphatics and distribute throughout the body. Morbidity associated with infection stems from both the persistence of the worm in the skin and the lymphatics that creates scarring and fluid accumulation and the MF causing debilitating chronic dermatitis due to irritation of MF antigen [3]. Resident within W. bancrofti and B. malayi parasites at all stages of life are the endosymbiotic bacteria, Wolbachia. These obligate intracellular bacteria are members of the Rickettsialaes order of α-proteobacteria [4] that also contain the mammalian pathogens in the genera Rickettsia, Brucella, Bartonella, Ehrlichia and

Anaplasma [5]. Typically, Wolbachia are present within hypodermal cells of the lateral cords of both sexes, however, vertical transmission of the bacteria to MF is facilitated by

51 the presence of bacteria in the ovaries, oocytes and embryos during development in females [6].

Within the last decade increasing concerns have arisen regarding the ability to effectively control and eradicate current infections of the filarial endoparasitic worms that cause onchocerciasis and LF [7]. It is estimated that 120 million people are currently suffering from LF and more than one billion individuals in 73 countries are at risk of developing LF [8]. In 2000, the WHO initiated a call to eliminate LF as a public-health problem by 2020 [9]. Significant steps have been taken towards this eradication effort but have been mainly limited to the use of mass drug administration of a microfilaricide to interrupt transmission and diminish morbidity. This approach has reduced the reoccurrence in some countries by 46% over the last 13 years [2, 9]. Drugs with the greatest efficacy toward LF have been limited to trials of annual, bi-annual, single-dose, multiple dose, and combinations of either ivermectin or diethylcarbamazine with albendazole [7, 10]. Recently, antimicrobial treatments such as doxycycline have been added to antifilarial regimens to target the symbiotic intracellular bacteria Wolbachia [11-

13]. Anti-Wolbachia drugs have been shown to reduce the pathogenicity and reproductive capacity of adult filarial worms [14]. The limitations associated with the use of the above drugs are four-fold: specific restrictions on patient age and health status due to cytotoxicity; the inability to reach the deep tissues where the adult filarial nematodes reside; counter-indications in areas endemic for the Loa loa parasite; and poor patient compliance [15]. These limitations have created an urgent need to seek new technologies that can be used to dramatically improve therapy protocols.

In the parasite, Wolbachia are vertically transferred from the ovaries of the adult into embryos that gestate and are shed as MF into the surrounding tissue [16]. In the adult female, the bacteria contribute to reproduction and embryo development. Treating adult worms in vitro and in vivo with tetracyclines, including doxycycline, to eliminate

Wolbachia has been shown to halt embryogenesis, reduce the amount and viability of MF shed [14, 17, 18] in tissues, and lead to death of the adult worm in 6 to 12 months [12,

19]. Loss of viability in the adult worm through anti-Wolbachia therapy is precipitated by loss of the endosymbiont, triggering apoptosis and tissue disruption [20]. Another antibiotic that is effective against Rickettsiae is rifampicin, which is better tolerated in children and pregnant patients, is able to kill Wolbachia in Onchocerca parasites in vitro

[21]. However, rifampicin is less effective than doxycycline in vivo [18, 22]. Additional therapeutic options for doxycycline are to combine it with other current antiparasitic drugs; however combination therapies present difficulties for drugs that have different half-lives, for widespread drug administration programs that suffer from poor patient compliance, and for long treatment regimens that require multiple years of therapy [23].

Amphiphilic polyanhydride nanoparticles have been studied extensively as carriers for drugs and vaccines [24-28]. These biodegradable materials degrade into dicarboxylic acids upon scission of the anhydride bond, rendering them highly biocompatible. These materials are generally hydrophobic and this confers a surface erosion mechanism to devices made of polyanhydrides [29, 30]. The advantage of such a surface erosion mechanism is that payloads can be released in a sustained and predictable manner, providing a so-called zero order release profile, in which the release rate of the payload is constant. Such release profiles enable single dose therapies, maximize the time

53 over which the in vivo drug concentration is maintained within the therapeutic window, and enhance patient compliance. This nanoscale platform also has the capability to be a delivery system for combination therapies that carry payloads to hard to reach tissues, thereby offering the potential to increase the efficacy of the drug-nematode interaction.

The ability of the polyanhydride nanoparticles to slowly erode and release the cargo molecule in a controlled manner can also allow for specificity against both the adult nematode and Wolbachia [11]. In this work, we demonstrate that co-delivery of doxycycline and ivermectin through encapsulation into polyanhydride nanoparticles effectively kills adult B. malayi filarial worms 8-fold faster with up to a 4,000-fold reduction in the amount of drug used. We hypothesize that the mechanism behind this enhanced killing of the macrofilaria is the ability of the nanoparticles to penetrate the outer membrane of the B. malayi worm and effectively deliver drug directly to the worm and its symbiotic bacteria Wolbachia at high enough microenvironment concentrations to cause death. The approach described herein holds great promise to interrupt the life cycle of the nematode not only by reducing the number of MF, but also by improving macrofilaricidal activity. These findings have the potential to dramatically enhance the treatment of patients suffering from debilitating filarial diseases such as LF and RB.

Materials and methods

Acquisition and storage of B. malayi and microfilariae

Live adult B. malayi females, males, and microfilariae were acquired from the

NIH/NIAID Infectious Disease Filariasis Research Reagent Repository Center (FR3) at the University of Georgia (Athens, GA). Adult worms were maintained in non-phenol red

Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-

54 inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin. The B. malayi were held in an incubator at a temperature of 37˚C supplemented with 5% carbon dioxide

(CO2). Female and male worms were stored individually in 48 well microtiter plates containing 1 mL of RPMI-1640. Previously shed microfilariae were housed in 50 mL conical tubes containing 25 mL of RPMI-1640. Upon arrival of the filarial worms they were separated and placed into individual 48 well microtiter plates that contained the previously described media.

Motility scoring

The motility of the adult worms treated with various formulations was observed for 30 seconds by utilizing a 2X objective on a Nikon Microscope and scored utilizing the following 0-5 scoring system: zero percent motility reduction, head and tail uninhibited as well as mid-section unaffected = 5; 1 to 25% motility reduction, ability to visualize both the head and tail movements easily as well as midsection = 4; 26 to 49% motility reduction, showing a partial mid-section paralysis = 3; 50 to 74% motility reduction, showing full mid-section paralysis followed by a substantial reduced movement in the head and tail = 2; 75 to 99% motility reduction, showing full mid- section paralysis followed by either head and or tail paralysis but not limited to occasional movement over a 30 second time period = 1; and a score of 0 represented

100% death (i.e., non-motile). The loss of motility of the adult worms was compared with that of respective untreated controls.

Synthesis of ivermectin and doxycycline-loaded polyanhydride nanoparticles Materials

For the synthesis of the 1,6-bis(p-carboxyphenoxy)hexane (CPH) and 1,8-bis(p- carboxyphenoxy)-3,6-dioxaoctane (CPTEG) monomers and the corresponding 20:80

CPTEG:CPH copolymer, and the ivermectin and doxycycline-loaded nanoparticles, acetic acid, acetic anhydride, acetone, acetonitrile, chloroform, dimethyl formamide, ethyl ether, hexane, methylene chloride, pentane, petroleum ether, potassium carbonate, sodium hydroxide, sulfuric acid, and toluene were purchased from Fisher Scientific (Fair- lawn, NJ). The chemicals 1,6-dibromohexane, 1-methyl-2-pyrrolidinone, hydroxybenzoic acid, N,N-dimethylacetamide, sebacic acid, and tri-ethylene glycol were obtained from

Sigma Aldrich (St. Louis, MO). The chemical 4-p-fluorobenzonitrile was purchased from

Apollo Scientific (Cheshire, UK). For 1H NMR analysis deuterated chloroform and deuterated dimethyl sulfoxide were purchased from Cambridge Isotope Laboratories (Ando- ver, MA). Rhodamine B, a fluorescent dye, and the drugs, ivermectin and doxycycline, were purchased from Sigma Life Science (St. Louis, MO).

Polymer synthesis

Synthesis of the CPH and CPTEG monomers was performed as described elsewhere [34]. Copolymers containing 20:80 molar ratios of CPTEG and CPH were synthesized through melt condensation, as previously described [28, 29]. Molecular weight and copolymer composition were confirmed using 1H NMR.

Nanoparticle synthesis

Doxycycline- and ivermectin-loaded 20:80 CPTEG:CPH nanoparticles were synthesized using solid/oil/oil nanoprecipitation, as previously described [25, 31, 32].

Briefly, ivermectin and doxycycline, each at 5% (w/w), and rhodamine B at 2% (w/w) of the total weight of the polymer were dissolved in methylene chloride at a concentration of

20 mg/mL followed by rapid precipitation into the anti-solvent, pentane. The polyanhydride nanoparticles were then collected using vacuum filtration. The drug-loaded particles were characterized for size and morphology by scanning electron microscopy (SEM,

FEI Quanta SEM, Hillsboro, OR). So, every 100 mg of the final nanoparticle preparation would contain 5 mg of IVM, 5 mg of doxycycline and 2 mg of rhodamine.

Drug release kinetics

The in vitro release kinetics of ivermectin and doxycycline from 20:80

CPTEG:CPH nanoparticles was determined using high performance liquid chromatography (HPLC). Nanoparticles were suspended in 1 mL of phosphate buffered saline

(PBS, pH 7.2), followed by thorough sonication to suspend the particles and incubated at

37°C under constant shaking. At each time point, the tubes were centrifuged at 10,000 rcf for 10 min. The supernatant was removed and replaced with fresh PBS and returned to incubation.

The ivermectin was separated using a Zorbax C8 4.6 x 250 cm chromatography column using a mobile phase of tetrahydrofuran, acetonitrile, and water in a 40:40:20 volume ratio, respectively. The flow rate was set at 1 mL/min and ivermectin eluted at a retention time of 5.3 min. The ivermectin was quantified with fluorescent detection using

57 an excitation wavelength of 365 nm and an emission wavelength of 475 nm with a gain of 10 and attenuation of 64 [33]. The doxycycline was quantified using a Varian Pursuit

XRs 3 C18 150 x 2 mm column. The mobile phase consisted of water with 0.1% formic acid and acetonitrile at a 98:2 volume ratio and at a flow rate of 0.2 mL/min. After one minute the mobile phase was ramped to 100% acetonitrile over 4 min. Doxycycline was quantified with tandem mass spectroscopy (MS/MS) using the following conditions: Q1 to Q3 transitions of 445 to 154 at 30 V collision energy and 445 to 427.9 at 16 V collision energy [34]. The encapsulation efficiency of the particles was determined as described previously [35] and found to be approx. 100% for both drugs and for the rhodamine.

Drug treatments of B. malayi

Ivermectin (22,23-dihydroavermectin B1) and doxycycline hyclate (C22H24N2O8 ·

HCl · 0.5H2O · 0.5C2H6O) were dissolved in DMSO at a final concentration of 0.02 % v/v. RPMI-1640 medium was prepared such that the final concentrations of each drug in nanoparticles were 195, 49, 10, 5, 1.95, 0.049, 0.01, 0.005, and 0.001 µM, respectively.

Control medium contained 0.02% DMSO and rhodamine B, but no drug. Individual worms that were previously placed into 48 well flat bottom culture plates upon arrival had new fresh media that contained 1 mL of RPMI-1640, 0.01% streptomycin and penicillin and 10% heat-inactivated fetal calf serum. B. malayi were incubated at 37̊ C and 5% CO2 for 1-2 hours for acclimatization to evaluate base line motility before the experiment began. The nanoparticle-encapsulated drugs and standard antifilarials (i.e., free drug with no nanoparticle encapsulation) were added as previously described above.

For example, 2 mg of the 195 µM drug concentration nanoparticles were added to each well and correspondingly lower amounts of nanoparticles were added as the drug

58 concentrations decreased. Controls received equal amounts of media but lacked the nanoparticles and standard antifilarials. B. malayi were incubated at 37̊C and 5% CO2 and monitored for 14 days post treatment.

Viability assessment

Reversibility assays where conducted 24 h following a 9 day course of treatment and or after the final individual within the soluble group reached a motility score of 0.

Individual worms where transferred to a new 48 well microtiter plate that contained 1 mL of the previously described media and were placed into an incubator at a temperature of

37˚C supplemented with 5% carbon dioxide (CO2). After 24 h a motility score was recorded. Upon completion of reversibility adult female worm viability was assessed utilizing a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

(MTT)-formazan colorimetric assay. The same female worms used in the motility assay were gently blotted and transferred to a 96 well microtiter plate that contained 0.2 mL of

1mg/mL MTT in phosphate-buffered saline (pH 7.2) and incubated for 2 h at 37°C. The formazan formed was extracted in 0.15 mL of DMSO for 4 h at 23°C and the absorbance was measured at 570 nm using a spectrophotometer (FLUOstar Omega version 1.01).

The mean absorbance of the formazan from the treated worms was compared with that of the controls. The viability of the treated worms was assessed by calculating the percent inhibition in motility and MTT reduction over the DMSO-treated control worms.

MF shedding and motility

The effect of treatments on MF shedding and subsequent motility was assessed using two experimental approaches. The direct effect on MF was performed by collecting

MF from the spent media of healthy untreated female worms. Spent media was centrifuged for 10 min at 200 rcf, the MF were re-suspended in fresh media and enumerated by counting using a 14.80 mm² grid plate. Aliquots containing at least 250

MF were dispensed into wells of a 96 well plate and treated in triplicate with either soluble drug, blank nanoparticles or nanoparticles containing drugs as indicated. Wells were then monitored and scored daily for MF movement. Motility of MF scoring was carried out using a 10X Nikon microscope and individual scores from five separate fields of view (FOV) were averaged to yield a motility score for each well. Scoring was adapted from a previously reported method by observing the movements of the anterior and posterior body over one min [36]. Data were analyzed by ANOVA and graphs were assembled using GraphPad Prism (v6.05). In separate experiments, the effect of treating adult female Brugia and their ability to release MF was monitored and recorded for 14 days. The motility of the shed MF was also recorded. Each experiment, the various treatment groups, and the different doses of the drugs were repeated for a minimum of three times.

Nanoparticle uptake by B. malayi

To understand the interaction between the nanoparticles and the filarial worms, recently deceased adult worms (motility score of “0”) from treated and untreated experimental groups were fixed and prepared for confocal microscopy. Worms were removed from treatment wells, washed twice in pH 7.4 PBS and fixed with 4%

60 paraformaldehyde for 1 h at room temperature. Worms were removed, rinsed with fresh

PBS and mounted carefully onto glass slides using Prolong Gold with DAPI (Life

Technologies). Slides were then stored in the dark at 4˚C prior to imaging. Imaging was performed using an Olympus IX-71 laser-scanning confocal microscope (Fluoview 2000, v2.0) equipped with 405 nm, 488 nm, 559 nm and 635 nm lasers, spectral photomultiplier detection and a piezo-controlled stage. Laser power and exposure settings were set according to the negative (worms with no rhodamine) and positive (rhodamine containing nanoparticles) and maintained constant over all the experiments. Image stacks were collected simultaneously for bright field (transmitted light - grey scale), cell nuclei (DAPI

- blue) and nanoparticles (rhodamine - red). Bright field and cell nuclei were used to identify various tissues, organs and structures in the worm such as oral opening, pharynx, uterus, the nerve ring, extracellular secretory apparatus, esophageal track and anal pore.

Rendering of Z-stack data in three dimensions and minimal image processing was performed as described previously by our laboratory [37] using ImageJ v 1.49a (NIH).

Software parameters for image processing remained constant for all image/data sets presented.

Results

Encapsulation of doxycycline and ivermectin into polyanhydride nanoparticles provides sustained drug release

After encapsulation of the anti-filarial drugs by nanoprecipitation, the particle morphology and size were examined using scanning electron microscopy. Surface morphology was found to be consistent with previous work and the scanning electron photomicrographs are shown in Figure 2-1 (panel B) [26, 27, 38]. Additionally, particle

61 size was also found to be consistent with previous work [37, 39]. The mean diameter of the drug-containing 20:80 CPTEG:CPH nanoparticles was 218 ± 56 nm.

Figure 3-1 Release kinetics of ivermectin (IVM) and doxycycline (DOX) Panel A: Release kinetics of ivermectin (IVM) and doxycycline (DOX) from 20:80 CPTEG:CPH nanoparticles quantified using HPLC. Panel B: Scanning electron photomicrograph of 20:80 CPTEG:CPH nanoparticles loaded with 5% IVM, 5% DOX, and 2% rhodamine. Scale bar: 500 nm.

Using a HPLC assay as described in the Methods section, the release kinetics of ivermectin and doxycycline from the nanoparticles was quantified, as shown in Figure 3-

1, panel A). The 20:80 CPTEG:CPH nanoparticle formulation provided sustained release of both drugs over one week. That data showed that the doxycycline release profile from the hydrophobic 20:80 CPTEG:CPH nanoparticles showed a small burst effect, which is consistent with previous work [40]. It was also observed that there was a much larger burst release of doxycycline as compared to that of ivermectin.

Polyanhydride nanoparticle-based drug delivery significantly increased B. malayi macrofilaria mortality and significantly reduced microfilarial shedding

The effectiveness of the polyanhydride nanoparticle-based delivery platform was compared to that of the standard soluble treatment in terms of the overall percent survival of B. malayi over the duration of the study with a single treatment of the previously described groups (Figures 3-2 and 3-3, panel A). The effect of blank (i.e., no drug) nanoparticles containing only rhodamine was assessed with 2 mg/mL of nanoparticles, the amount corresponding to that of the highest drug concentration administered (i.e., 195

µM). Similarly, untreated controls did not contain active drugs, but were supplemented with rhodamine and DMSO corresponding to the amounts present in the highest treatment group (i.e., 195 µM drug concentration).

The motility of the worms treated with the blank nanoparticles did not vary significantly from that of the untreated worms for either female or male worms (Figures

3-2 and 3-3 and Supplemental Figure 1). The results clearly demonstrate the effectiveness of the nanoparticle formulations for all the treatment groups when compared to the soluble treatment group for drug concentrations as low as 1 nM. Another parameter to quantify the difference between the effectiveness of the soluble dual ivermectin/doxycycline treatment and that of the nanoparticle-based delivery platform was the average number of days to death. We conducted a fourteen-day in vitro study that quantified the average days to death of female (Figure 3-2, panel B) and male (Figure 3-

3, panel B) B. malayi. Random testing of viability of worms exhibiting a motility score of

0 for metabolic activity was performed using the MTT reduction assay throughout the

63 studies and for all treatment groups (data not shown) and a representative of % MTT inhibition analysis is shown in Supplemental Figure 3-2 for the 5 µM drug concentration.

Inhibition of metabolic activity greater than 75% of that of untreated controls indicated that worms were non-viable and declared dead. In addition, separate worms with a motility score of 0 were subjected to reversibility testing whereby after 24 h of no motility, worms were removed from the test media, washed three times and placed in fresh media with no drugs (data not shown). Recovery of movement was interpreted as the worms being temporarily paralyzed and those worms were removed from the ATD, except where indicated† (Figures 3-2 and 3-3, panel A). We observed reversible paralysis only when high doses of soluble drug (49 µM - 195 µM) were used in approximately

25% of all worms treated in these groups. Strikingly, reversible paralysis was not observed in any nanoparticle-treated group of any drug concentration, showing that the nanoparticle formulations effectively killed the worms. The data indicated that the nanoparticle-based delivery platform significantly (p<0.001) lowered the overall time to death when compared directly to the soluble dual ivermectin/doxycycline treatment. Treatment of female worms with 195 µM of the soluble drugs resulted in an average time of death in excess of nine days and 63% of the worms were killed (Figure 3-2, panels A and B). In contrast, treating the worms with the nanoparticle-based delivery system with the same drug concentration sharply reduced the average time to death to less than 1.2 days, and more significantly, 100% of the worms were killed. We also performed dose titration studies and systematically lowered the concentration of the two drugs from 195 µM to 1 nM.

Figure 3-2 Panel A. Table outlining survival of B. malayi females after treatment with 195, 49, 10, 5, 1.95, or 0.049 µM concentrations of ivermectin (IVM) and doxycycline (DOX) delivered solubly or encapsulated into 20:80 CPTEG:CPH nanoparticles. Panel B. Average number of days to death of B. malayi females after administration of soluble or encapsulated IVM/DOX treatments and comparison to control worms. Significance was determined at p<0.05, 0.01, or 0.001 as noted using a Student’s T test. Panel C. Average motility scores of B. malayi females after IVM/DOX treatments scored using a 2X objective on a Nikon Microscope following a 0-5 scoring system, as described in the Methods. Panel D. Average number of microfilaria shed by B. malayi females after administration of soluble or encapsulated IVM/DOX treatments and comparison to control worms. The NP only group contains comparable amount of rhodamine and the total amount of particle in this group corresponds to that of the highest drug concentration of 195 µM. At 14 days, all worms treated with NP only with a motility score of 0 remained viable based on the MTT assay and recovery of motility upon transferring to fresh medium†. Significance was determined at p<0.05, 0.01, or 0.001 as noted using a Student’s T test.

Female worms treated with the 1 nM concentration of soluble ivermectin/doxycycline resulted in an average time to death that was >14 days, and more significantly, only 33% of the worms were killed at this concentration (Figure 3-2, panel

A).In contrast, the worms treated with the nanoparticle-based delivery system containing the 1 nM drug concentration had an average time to death of <6 days, and more significantly, 100% macrofilarial death was observed upon treatment with the low concentration of ivermectin/doxycycline.

To determine if nanoparticles changed the reproductive capacity of female worms the shedding of MF was observed for fourteen days (Figure 3-2, panel D). Both the soluble ivermectin/doxycycline and the nanoparticle-based delivery groups significantly reduced the overall microfilariae shed at the high drug concentration of 195 µM. But as the concentration of ivermectin/doxycycline was systematically reduced to 5 nM, significant differences (p<0.01) were observed in the MF shed by the worms treated with the soluble ivermectin/doxycycline groups compared to the worms that were treated with the nanoparticle-based delivery system, with the nanoparticles being more effective at lower drug concentrations. The effect of the various drug treatments on the motility of B. malayi MF was observed for 14 days (Figure 3-4). Similar to the effects observed for adults, the average time to death as well as the motility of the MF treated with the soluble drug was significantly (p < 0.001) higher in comparison to that of the MF that were treated with the nanoparticle-based delivery system, especially at low drug concentrations.

For the B. malayi males (Figure 3-3, panels A and B) treatment with drug-loaded

20:80 CPTEG:CPH nanoparticles showed a significant difference (p<0.05) in terms of

66 survival when compared to the survival of worms treated with an equivalent soluble dose of ivermectin/doxycycline. In terms of average time to death, male worms treated with a

195 µM concentration of soluble ivermectin/doxycycline died after seven days and more significantly, only 30% of them were killed at this dose (Figure 3-3, panel A).

Figure 3-3 Panel A. Table outlining survival of B. malayi males after treatment with 195, 49, 10, 5, 1.95, or 0.049 µM concentrations of ivermectin (IVM) and doxycycline (DOX) delivered solubly or encapsulated into 20:80 CPTEG:CPH nanoparticles. Panel B. Average number of days to death of B. malayi males after soluble or encapsulated IVM/DOX treatments as compared to control worms. Significance was determined at p<0.05, 0.01, or 0.001 as noted using a Student’s T test. Panel C. Average motility scores of B. malayi males after IVM/DOX treatments scored using a 2X objective on a Nikon Microscope following a 0-5 scoring system, as described in the methods.

In sharp contrast, all the male worms treated with the nanoparticle-based delivery system containing the same dose of ivermectin/doxycycline died in less than one day.

When the treatment concentration was reduced to 0.049 µM, the average time to death of worms treated with soluble ivermectin/doxycycline was >14 days with a death rate of only 20%. On the other hand, all the male worms treated with the nanoparticle-based delivery system containing the 0.049 μM concentration of Ivermectin/doxycycline died within ten days.

Figure 3-4 The motility of B. malayi MF treated with decreasing doses of either soluble or encapsulated ivermectin (IVM)/doxycycline (DOX) was recorded for 14 days post treatment. The recorded motility scores (Panel B) were used to calculate an average time to death for each treatment group and dose (Panel A). To calculate the average motility score for each time, dose and treatment group, triplicate wells containing a minimum of 200 MF in each well were treated as indicated. A motility score of 0 was equated with death and the average time to death was plotted along with standard error. Data presented are from one of two experiments with similar results. Significance was determined at p<0.05, 0.01, or 0.001 as noted using a Student’s T test.

In conjunction with the average time to death assay we also monitored the motility over time to characterize the overall effectiveness of the nanoparticle treatments

(panel C of Figures 3-2 and 3-3). With both male and female B. malayi worms, an exponential reduction in overall motility was observed in the worms administered the nanoparticle-based delivery treatments in comparison to that of the worms that received the soluble drug treatment. system containing the 0.049 µM concentration of ivermectin/doxycycline died within ten days

Polyanhydride nanoparticle-based treatment effectively penetrated the worm cuticle

To visualize the interactions of polyanhydride nanoparticles with B. malayi female worms, animals were treated with equivalent amounts of a cocktail containing rhodamine red dye with ivermectin/doxycycline either in soluble form or encapsulated within the 20:80 CPTEG:CPH nanoparticles. At regular intervals worms were washed three times in PBS to remove surface-bound nanoparticles, fixed, and then imaged using confocal microscopy to localize nanoparticles within tissues. Detection of focal, intense rhodamine staining is consistent with size and staining patterns of intact nanoparticles. In comparison, diffuse red staining is indicative of free rhodamine. Worms treated with 195

µM of the soluble drug cocktail did not accumulate rhodamine within tissues over a period of 96 hours (Figure 3-5, panel A).

Over this same time interval, the much lower dose of 5 µM of ivermectin/doxycycline encapsulated within the nanoparticles that also contained rhodamine, extensively labeled the interior structures of female worms (Figure 3-5, panel

B). Diffuse red staining reveals the rapid distribution of the released dye throughout the-

Figure 3-5 Confocal microscopy of female B. malayi with nanoparticles. Worms were incubated for 96 h with either soluble controls (panel A) or 20:80 CPTEG:CPH nanoparticles containing ivermectin (IVM), doxycycline (DOX), and rhodamine B (panel B), fixed and imaged by LSCM. Controls contained 195 µM each of IVM and DOX, and 3.9 µM rhodamine B, while the nanoparticles contained 5 µM of each drug and 0.1 µM of rhodamine B. Left panels are DNA (blue), rhodamine (red) and bright field image overlays and right panels are the respective individual rhodamine images collected using identical image acquisition settings. Inset box within the nanoparticle-treated worm outlines the area selected for side view rendering (C). Representative images shown demonstrate the accumulation of nanoparticles (arrows) within tissues throughout the worm (B) as compared to the higher amount of soluble rhodamine that was not detected within the body of the worms. Similar results were seen throughout the body of the worm in both male and female worms (data not shown).

-body of the worm and focal, intact 20:80 CPTEG:CPH nanoparticles are easily discerna- ble deep within worm tissues by confocal microscopy (Figure 3-5, panels B and C).

Three dimensional reconstruction of confocal Z-stacked images reveal the penetration of

70 nanoparticles and release of rhodamine throughout the inner tissues of the worm (Figure

3-5, panel C). Similar rhodamine and nanoparticle staining patterns were evident throughout the length of the treated worms at all concentrations tested. Internal staining of worms treated five hours post treatment with 195 µM of the soluble drug cocktail were identical to the rhodamine intensity of worms treated with 5 µM of the drug cocktail encapsulated within the nanoparticles for 96 hours. These observations offer insights into why the nanoparticle-based delivery treatments of B. malayi are highly effective, especially at low doses.

Discussion

The World Health Organization global program to eliminate LF recommends annual dosing of albendazole with either ivermectin or diethylcarbamazine to be continued for at least five years. These drugs are potent antihelmintics that kill and reduce shedding of MF. However, they exhibit little direct activity against the adult filarial worms [41]. Discovery of the endosymbiotic bacteria Wolbachia and the critical role they play in the biology and viability of the adult worm revealed a potential target that could be exploited to improve therapies for RB and LF [19, 21, 42]. The addition of anti-bacterials (such as doxycycline or tetracycline) to the treatment regimen can reduce parasite embryogenesis [20, 43], improve macrofilaricidal activity, and reduce hydrocele pathology in the host [44, 45]. To achieve maximal benefit, daily dosing of 200 mg for 3-

5 weeks of antibacterial drug is required and can be a difficult barrier to overcome for

Mass Drug Administration (MDA) programs [15]. In this work, we report that the use of a polyanhydride nanoparticle-based carrier effectively reduced the amount of drug

71 necessary for macrofilarial death by up to 4,000-fold compared to physiologically relevant doses.

The unique chemistry of the polyanhydride nanoparticles helps address many of the challenges associated with mass drug administration against lymphatic filariasis. The surface erosion profile of the polyanhydride nanoparticles sustains the slow release of drug at different kinetics for both drugs from the same particle as shown in Figure 3-1.

The initial burst of the doxycycline released from the 20:80 CPTEG:CPH nanoparticles is faster than that observed for ivermectin. Previous work has shown that this copolymer composition contains weakly segregated CPTEG- and CPH-rich domains [46]. This may indicate a preferential partitioning of the hydrophobic ivermectin to the slowly degrading

CPH-rich domains of the copolymer, leading to the slow release profile observed [35,

47]. In contrast, the less hydrophobic doxycycline is released faster, suggesting partitioning into the faster eroding CPTEG-rich domains. This controlled release profile of the drugs from the polyanhydride nanoparticles may explain the enhanced efficacy observed with the nano-carrier treatments. The payload is protected from an aqueous environment for longer periods of time, which may prevent degradation of the drugs, leading to the reduction in required dose observed in this work. Additionally, based on the confocal microscopy studies described in Figure 3-5, it appears that these nanoparticles effectively interact with the worm cuticle and behave like a Trojan horse in terms of transporting the drug cocktail into the worm, bypassing excretory pumps.

Together, the encapsulation and subsequent controlled release of the drugs at high concentrations both internally to the worm and localized to the cuticle may account for the enhanced killing observed in Figures 3-2 and 3-3.

Reducing the effective amount of drug required would have several benefits on improving therapeutic treatment of filarial diseases. We predict that increasing drug effectiveness would likely reduce unwanted side effects by reducing amount of drug needed and contribute to improving patient compliance [43, 48, 49]. As discussed above, we hypothesize that encapsulation and subsequent controlled release of the drug and the ability of the polyanhydride nanoparticle carriers to localize to the worm cuticle surface, and even be internalized by the worms, lead to a higher microenvironment concentration of the drugs. In contrast, the soluble drug cocktail is a liquid, and so to create the same local concentration internally to the worm and at the cuticle surface, more drug must be administered initially. This is supported by the confocal microscopy data in Figure 3-5, which show that the polyanhydride nanoparticles readily localize to the worm, increasing the concentration of the internalized dye as compared to the soluble control. In vivo, while the soluble drug will be rapidly cleared from the body, the nanoparticle- encapsulated drug will persist in different tissues for periods up to 28 days, as shown by our previous studies [50, 51]. Another difficulty in treating filarial infections is that individuals are continually being exposed to new parasites even after successful completion of antiparasitic regimen [52]. It was reported by Walker et al. that while standard doxycycline therapy was able to reduce the majority of macrofilaria in human subjects, reinfection with drug-naïve parasites occurred [19]. In this scenario, we would predict that the delayed release capacity of the nanoparticles would be able to extend the effective range beyond the time frame afforded by standard dosing.

The polyanhydride nanoparticle drug delivery platform decreased the average time to death of B. malayi females and facilitated a rapid decrease in the motility of

73 treated worms as shown in Figure 3-2, even at nanomolar concentrations of the drugs.

Similar trends were also observed in the B. malayi males to a slightly lesser extent, possibly reflecting the comparatively lower number of Wolbachia in male compared to female macrofilariae (Figure 3-3). Rapid killing would likely contribute to the dramatic reduction in shedding by female worms at low nanoparticle concentrations (Figure 3-2, panel D). Effectiveness at the lower concentrations may prove to be more beneficial as rapid killing of MF is known to induce severe side-effects within the host [53]. It is also likely that the nanoparticles increased the rate of delivery of ivermectin and rapidly paralyzed the muscles within the body of the worm and the vagina, which are needed to expel the MF. A similar in vitro observation was made by Tompkins et al. with Brugia treated with either moxidectin or ivermectin [41].

Finally, the confocal microscopy data in Figure 3-5 provides an initial explanation of the strong interactions between polyanhydride nanoparticles and the cuticle surface, which is hypothesized to lead to the efficacy profile of this nano-carrier platform.

Tracking the fluorescent rhodamine B co-loaded into the nanoparticles demonstrated a greater increased influx of payload into the worm compared to drug and dye delivered solubly. As previously stated, in addition to providing sustained release of drug, with implications for dose sparing and patient compliance, the drug-loaded polyanhydride nanoparticles demonstrated enhanced internalization by the B. malayi worms. One of the benefits of this type of Trojan horse behavior of the nanoparticles is the ability to create microenvironments with high drug concentrations, in contrast to soluble drug cocktail treatments, which diffuse in an aqueous solution [54]. To achieve the benefit of an increased local drug concentration, the nanoparticles must interact with the target cell, or,

74 in this case, the B. malayi adult worm or microfilaria. The data in Figure 3-5 clearly showed that the nanoparticles are interacting with the worms in a way not previously described, improving the rapid delivery of drugs. We speculate that the nanoparticles undergo facilitated diffusion into the worm, by crossing the cuticle, penetrating the hypodermis membrane barriers and accessing deeper tissues within the worm [55, 56].

Preliminary assessment of treated worms by dissection corroborates cuticular penetration because far fewer nanoparticles were observed in the gut and pharynx of worms, while they did accumulate in deeper tissues including the hypodermis. More detailed studies are underway in our laboratories to further delineate the mechanisms of nanoparticle transport across the cuticle of the worms.

Resistance to anthelmintic drugs is on the rise due in part to the over-expression of ABC-transporters that act as multi-drug resistant (MDR) efflux pumps in many parasites, including Onchocerca and Brugia [57]. Our success with delivery of drug cocktails within nanoparticles needs to be analyzed in the context of previous work, wherein the approach of delivering drug combinations was shown to reduce the development of resistance in parasites [18]. However, adoption of cocktail therapy into

MDA programs remains problematic due to the need to maintain high levels of doxycycline [18]. We posit that encapsulating low doses of these drugs into the Trojan horse polyanhydride nanoparticles address the difficulties in maintaining prolonged delivery of antiparasitic drugs. The activity of the efflux pumps would normally reduce effective drug concentrations within the worm by preventing absorption through the esophagus as well as reducing intracellular concentration within cells. We speculate based on our results that the efficient and rapid direct penetration of the polyanhydride

75 nanoparticles through the surface of the worm would bypass poor esophageal absorption.

We hypothesize that accessing the worm through this unique mechanism would be a selective pressure that would be difficult for the parasite to overcome and provide a paradigm-changing technology to combat filarial disease.

Conclusions

Filarial diseases represent a significant social and economic burden in areas that are endemic with the filarial endoparasite B. malayi, and its symbiotic bacteria

Wolbachia. We report the use of a polyanhydride nanoparticle-based drug delivery platform for the co-delivery of the antiparasitic drug, ivermectin, to reduce macro and microfilarial burden and the antimicrobial, doxycycline, to eliminate the symbiotic

Wolbachia. The co-delivery of doxycycline and ivermectin in the context of polyanhydride nanoparticles effectively killed adult female and male B. malayi filarial worms with up to a 4,000-fold reduction in the amount of drug used. Further, the time to death of the macrofilaria was significantly reduced when the anti-filarial drug cocktail was delivered in the context of the polyanhydride nanoparticles. Confocal microscopy experiments suggest that the polyanhydride nanoparticles behave like a Trojan horse and penetrate the outer membrane of the worm cuticle. The nanoparticles effectively deliver the drugs at high enough microenvironment concentrations to vital areas within the worm, thus significantly enhancing their effectiveness at killing the worms. These findings may have significant consequences for the reduction in the amount of drug and the length of treatment required for filarial infections and provide a paradigm-changing technology to combat filarial disease.

Acknowledgements

The authors gratefully acknowledge financial support from the Bill and Melinda

Gates Foundation Grand Challenges Exploration Opportunity (1087610) and the support of NIH, NIAID R01 AI047195. We also acknowledge the Filariasis Research Reagent

Resource Centre (FR3; College of Veterinary Medicine, University of Georgia, USA) for the supply of live adult Brugia. The content is solely the responsibility of the authors and does not necessarily represent the views of the National Institute of Allergy and

Infectious diseases. B.N. also acknowledges the Vlasta Klima Balloun Professorship. We would like to thank Dr. Judy Sakanari for technical assistance and critical reading of the revised manuscript. We would also like thank Dr. Saurabh Verma with technical assistance and interpretation with worm dissection.

Figure 3-6 Synergism of ivermectin (IVM) and doxycycline (DOX) in reducing motility of adult B. malayi females following encapsulation of drugs into nanoparticles. Average time to death of three worms treated in separate wells is shown in Panel B. Average motility scores of B. malayi females (Panel C) after treatments where visually scored with the 0-5 scoring system, as described in the methods. Panel D Indicates the use of the “Worminator”, a computer motion screening program utilized for motility scoring in B. malayi (Marcellino C, Gut J, Lim KC, Singh R, McKerrow J, & Sakanari J (2012). WormAssay: a novel computer application for whole-plate motion-based screening of macroscopic parasites. PLoS Negl Trop Dis 6(1): e1494). The percent dead were worms with a motility score of 0 (Panel A) and confirmed by MTT to show inhibition greater than 80% of untreated control

Figure 3-7 Enzymatic reduction of MTT reagent by adult B. malayi females following incubation with various treatments for 9 days. Motility of worms tested by the MTT assay is shown in Figure 3-6. Inhibition was calculated using optical density values of reduction of MTT reagent for untreated worms to represent maximum metabolic activity (Panel B). Changes in reduction of MTT recorded values for soluble and nanoparticle-based drug treatment groups as compared to untreated worms were calculated as percent inhibition (Panel A). Three individually treated worms were used for each treatment group and individual MTT percent inhibition was used to calculate the average and standard error. Significance difference of p<0.01 was calculated between the soluble IVM/DOX and the nanoparticle-encapsulated IVM/DOX. Inhibition greater than 75% was interpreted as loss in viability.

References

1. Ottesen, E.A., et al., The global programme to eliminate lymphatic filariasis: health impact after 8 years. PLoS Negl Trop Dis, 2008. 2(10): p. e317.

2. Coffeng, L.E., et al., African Programme For Onchocerciasis Control 1995-2015: model-estimated health impact and cost. PLoS Negl Trop Dis, 2013. 7(1): p. e2032.

3. Paily, K.P., S.L. Hoti, and P.K. Das, A review of the complexity of biology of lymphatic filarial parasites. J Parasit Dis, 2009. 33(1-2): p. 3-12.

4. Bandi, C., et al., Phylogeny of Wolbachia in filarial nematodes. Proc Biol Sci, 1998. 265(1413): p. 2407-13.

5. Bowman, D.D., Introduction to the alpha-proteobacteria: Wolbachia and Bartonella, Rickettsia, Brucella, Ehrlichia, and Anaplasma. Top Companion Anim Med, 2011. 26(4): p. 173-7.

6. Bouchery, T., et al., The symbiotic role of Wolbachia in Onchocercidae and its impact on filariasis. Clin Microbiol Infect, 2013. 19(2): p. 131-40.

7. Taylor, M.J., A. Hoerauf, and M. Bockarie, Lymphatic filariasis and onchocerciasis. Lancet, 2010. 376(9747): p. 1175-85.

8. Babu, S. and T.B. Nutman, Immunopathogenesis of lymphatic filarial disease. Semin Immunopathol, 2012. 34(6): p. 847-61.

9. Ichimori, K. Progress report 2000-2009 and strategic plan 2010-2020 of the global programme to eliminate lymphaticfi lariasis: halfway towards eliminating lymphatic fi lariasis. 2011. 10. Hoerauf, A., Filariasis: new drugs and new opportunities for lymphatic filariasis and onchocerciasis. Curr Opin Infect Dis, 2008. 21(6): p. 673-81.

11. Taylor, M.J., et al., Wolbachia bacteria of filarial nematodes: a target for control? Parasitol Today, 2000. 16(5): p. 179-80.

12. Langworthy, N.G., et al., Macrofilaricidal activity of tetracycline against the filarial nematode Onchocerca ochengi: elimination of Wolbachia precedes worm death and suggests a dependent relationship. Proc Biol Sci, 2000. 267(1448): p. 1063-9.

13. Bazzocchi, C., et al., Combined ivermectin and doxycycline treatment has microfilaricidal and adulticidal activity against Dirofilaria immitis in experimentally infected dogs. Int J Parasitol, 2008. 38(12): p. 1401-10.

14. Turner, J.D., et al., Macrofilaricidal activity after doxycycline only treatment of Onchocerca volvulus in an area of Loa loa co-endemicity: a randomized controlled trial. PLoS Negl Trop Dis, 2010. 4(4): p. e660.

15. Hoerauf, A., et al., Filariasis in Africa--treatment challenges and prospects. Clin Microbiol Infect, 2011. 17(7): p. 977-85.

16. Fischer, K., et al., Tissue and stage-specific distribution of Wolbachia in Brugia malayi. PLoS Negl Trop Dis, 2011. 5(5): p. e1174.

17. Sacchi, L., et al., Ultrastructural evidence of the degenerative events occurring during embryogenesis of the filarial nematode Brugia pahangi after tetracycline treatment. Parassitologia, 2003. 45(2): p. 89-96.

18. Hoerauf, A., et al., Efficacy of 5-week doxycycline treatment on adult Onchocerca volvulus. Parasitol Res, 2009. 104(2): p. 437-47.

19. Walker, M., et al., Therapeutic Efficacy and Macrofilaricidal Activity of Doxycycline for the Treatment of River Blindness. Clin Infect Dis, 2014.

20. Landmann, F., et al., Anti-filarial activity of antibiotic therapy is due to extensive apoptosis after Wolbachia depletion from filarial nematodes. PLoS Pathog, 2011. 7(11): p. e1002351.

21. Townson, S., et al., Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro. Filaria J, 2006. 5: p. 4.

22. Specht, S., et al., Efficacy of 2- and 4-week rifampicin treatment on the Wolbachia of Onchocerca volvulus. Parasitol Res, 2008. 103(6): p. 1303-9.

23. Tamarozzi, F., et al., Long term impact of large scale community-directed delivery of doxycycline for the treatment of onchocerciasis. Parasit Vectors, 2012. 5: p. 53.

24. Petersen, L.K., A.V. Chavez-Santoscoy, and B. Narasimhan, Combinatorial Synthesis of and high-throughput protein release from polymer film and nanoparticle libraries. J Vis Exp, 2012(67).

25. Ulery, B.D., et al., Design of a protective single-dose intranasal nanoparticle- based vaccine platform for respiratory infectious diseases. PLoS One, 2011. 6(3): p. e17642.

26. Petersen, L.K., C.K. Sackett, and B. Narasimhan, Novel, high throughput method to study in vitro protein release from polymer nanospheres. J Comb Chem, 2010. 12(1): p. 51-6.

27. Carrillo-Conde, B., et al., Encapsulation into amphiphilic polyanhydride microparticles stabilizes Yersinia pestis antigens. Acta Biomater, 2010. 6(8): p. 3110-9.

28. Kipper, M.J., et al., Design of an injectable system based on bioerodible polyanhydride microspheres for sustained drug delivery. Biomaterials, 2002. 23(22): p. 4405-12.

29. Torres, M.P., et al., Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. J Biomed Mater Res A, 2006. 76(1): p. 102-10.

30. von Burkersroda, F., L. Schedl, and A. Gopferich, Why degradable polymers undergo surface erosion or bulk erosion. Biomaterials, 2002. 23(21): p. 4221-31.

31. Haughney, S.L., et al., Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles. Acta Biomater, 2013. 9(9): p. 8262-71.

32. Huntimer, L., et al., Evaluation of biocompatibility and administration site reactogenicity of polyanhydride-particle-based platform for vaccine delivery. Adv Healthc Mater, 2013. 2(2): p. 369-78.

33. Prieto, J.G., et al., Improved LC method to determine ivermectin in plasma. J Pharm Biomed Anal, 2003. 31(4): p. 639-45.

34. Mastovska, K. and A.R. Lightfield, Streamlining methodology for the multiresidue analysis of beta-lactam antibiotics in bovine kidney using liquid chromatography-tandem mass spectrometry. J Chromatogr A, 2008. 1202(2): p. 118-23.

35. Shen, E., et al., Mechanistic relationships between polymer microstructure and drug release kinetics in bioerodible polyanhydrides. J Control Release, 2002. 82(1): p. 115-25.

36. Khunkitti, W., Y. Fujimaki, and Y. Aoki, In vitro antifilarial activity of extracts of the medicinal plant Cardiospermum halicacabum against Brugia pahangi. J Helminthol, 2000. 74(3): p. 241-6.

37. Ulery, B.D., et al., Polymer chemistry influences monocytic uptake of polyanhydride nanospheres. Pharm Res, 2009. 26(3): p. 683-90.

38. Petersen, L.K., et al., Amphiphilic polyanhydride nanoparticles stabilize Bacillus anthracis protective antigen. Mol Pharm, 2012. 9(4): p. 874-82.

39. Petersen, L.K., C.K. Sackett, and B. Narasimhan, High-throughput analysis of protein stability in polyanhydride nanoparticles. Acta Biomater, 2010. 6(10): p. 3873-81.

40. Carrillo-Conde, B.R., et al., Chemistry-dependent adsorption of serum proteins onto polyanhydride microparticles differentially influences dendritic cell uptake and activation. Acta Biomater, 2012. 8(10): p. 3618-28.

41. Tompkins, J.B., L.E. Stitt, and B.F. Ardelli, Brugia malayi: in vitro effects of ivermectin and moxidectin on adults and microfilariae. Exp Parasitol, 2010. 124(4): p. 394-402.

42. Slatko, B.E., M.J. Taylor, and J.M. Foster, The Wolbachia endosymbiont as an anti-filarial nematode target. Symbiosis, 2010. 51(1): p. 55-65.

43. Hoerauf, A., et al., Doxycycline in the treatment of human onchocerciasis: Kinetics of Wolbachia endobacteria reduction and of inhibition of embryogenesis in female Onchocerca worms. Microbes Infect, 2003. 5(4): p. 261-73.

44. Shakya, S., et al., Prior killing of intracellular bacteria Wolbachia reduces inflammatory reactions and improves antifilarial efficacy of diethylcarbamazine in rodent model of Brugia malayi. Parasitol Res, 2008. 102(5): p. 963-72.

45. Dangi, A., et al., Improvement in the antifilarial efficacy of doxycycline and rifampicin by combination therapy and drug delivery approach. J Drug Target, 2010. 18(5): p. 343-50.

46. Shen, E., et al., Microphase separation in bioerodible copolymers for drug delivery. Biomaterials, 2001. 22(3): p. 201-10.

47. Halley, B.A., W.J. VandenHeuvel, and P.G. Wislocki, Environmental effects of the usage of avermectins in livestock. Vet Parasitol, 1993. 48(1-4): p. 109-25.

48. Babalola, O.E., Ocular onchocerciasis: current management and future prospects. Clin Ophthalmol, 2011. 5: p. 1479-91.

49. Taylor, M.J., et al., Anti-Wolbachia drug discovery and development: safe macrofilaricides for onchocerciasis and lymphatic filariasis. Parasitology, 2014. 141(1): p. 119-27.

50. Petersen, L.K., et al., Combinatorial evaluation of in vivo distribution of polyanhydride particle-based platforms for vaccine delivery. Int J Nanomedicine, 2013. 8: p. 2213-25.

51. Haughney, S.L., et al., Effect of nanovaccine chemistry on humoral immune response kinetics and maturation. Nanoscale, 2014. 6(22): p. 13770-8.

52. Specht, S., et al., Newly acquired Onchocerca volvulus filariae after doxycycline treatment. Parasitol Res, 2009. 106(1): p. 23-31.

53. Zahner, H., Induction and prevention of shock-like lethal side effects after microfilaricidal treatment in filariae infected rodents. Trop Med Parasitol, 1995. 46(4): p. 221-9.

54. Davis, M.E., Z.G. Chen, and D.M. Shin, Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat Rev Drug Discov, 2008. 7(9): p. 771-82.

55. Devaney, E. and R.E. Howells, The microfilaricidal activity of ivermectin in vitro and in vivo. Tropenmed Parasitol, 1984. 35(1): p. 47-49.

56. Howells, R.E., A.M. Mendis, and P.G. Bray, The mechanisms of amino acid uptake by Brugia pahangi in vitro. Z Parasitenkd, 1983. 69(2): p. 247-53.

57. Ardelli, B.F., Transport proteins of the ABC systems superfamily and their role in drug action and resistance in nematodes. Parasitol Int, 2013. 62(6): p. 639-46.

CHAPTER 4 . NANOPARTICLE-BASED DRUG DELIVERY INCREASED SYNERGISM OF FIRST-LINE ANTI-FILARIAL DRUG ACTIVITY AGAINST BRUGIA PAHANGI

For publication in PLOS Neglected Tropical Diseases

Andrea M. Binnebose1, Adam S. Mullis2, Shannon L. Haughney2, Richard Martin3, Balaji Narasimhan2*, and Bryan H. Bellaire1* 1Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, 50011 2Chemical and Biological Engineering Iowa State University, Ames, 50011 3Biomedical Sciences Iowa State University, Ames, 50011

* To whom all correspondence must be addressed

E-mail: [email protected]; Phone: (262) 384-1086, [email protected]; Phone: (515) 294-1006, [email protected]; Phone:(515) 294-8019

Abstract

Amphiphilic polyanhydride nanoparticles (NP) are chemically and structurally distinct from other polymer or lipid-based particle delivery systems. These NPs are solid, surface eroding particles, which encapsulate small molecules or proteins that become an integral component of the particle. Their variable hydrophobicity imparts a unique, native cellular affinity leading to a high degree of internalization by mononuclear phagocytes

(MP). The reticular endothelial system consisting of dendritic cells, blood monocytes, and tissue-resident macrophages found in the liver, spleen, and lymph nodes that are

85 responsible for clearing, processing, and degrading foreign materials from circulation.

We sought to capitalize on two of the unique characteristics of NPs: extending intracellular persistence where the particles slowly erode and release the cargo molecule in a controlled manner. As well as, through the delivery of a co-encapsulated antiparasitic therapy consisting of albendazole, diethylcarbamazine or ivermectin along with the antibacterial drug doxycycline. In vitro studies with B. pahangi demonstrate an increase in antiparasitic activity, as measured by recording worm motility following exposure to soluble or encapsulated drugs. The combination of doxycycline with ivermectin for the average time of death (TOD) observed in B. pahangi females was reduced from 6.5 days for the soluble to 4 at the highest concentration shown (25 μg) of NPs. To achieve matched drug efficacy to that of the soluble drug, the overall NP concentration was reduced 500 times to achieve match antiparasitic activity. The use of NPs with the drug combination diethylcarbamazine/albendazole/doxycycline showed an even greater impact on parasitic killing when comparing the highest concentration (25 μg) from 9 days with soluble to 2 days for encapsulated drugs. As well as similar reductions in the necessary overall drug concentration as with the ivermectin groups. When comparing the lowest concentration of

NPs (0.05 µg) against the highest concentration (25 μg), the NPs were able to kill an additional three days faster against adult female worms. Similar results were obtained with adult B. pahangi male worms. We envision that encapsulation of drugs into NP will serve to repurpose current anti-filarial treatment methods into more potent and targeted therapies by offering increased patient compliance and increasing efficacy of the drugs.

Introduction

The parasitic helminth Wuchereria bancrofti, Brugia malayi, and B. timori are the causative agents of Lymphatic Filariasis (LF). Currently, over 120 million people are at risk of contracting Lymphatic Filariasis, with an average of 38.5 million people showing clinically active filariasis. While efforts to mitigate the effect of these diseases have been successful in some regions, current medications are insufficient to reach elimination by

2020 [1-3].The disease is characterized by fever, lymphangitis, hydrocele, lymphoedema, and elephantiasis, and is one of the most common causes of arthropod-mediated disability

[3, 4]. The unique parasitic life cycle begins when the larval microfilariae (MF) are aquired up during a blood meal from an infected host by the insect vector. The MF (L1) undergoes two larval molting’s stages L2, and the infective L3 stage, this takes around

10-14 days to occur [5]. When the infected mosquito feeds on another human host, the infective larvae (L3) are deposited within host tissue at the bite site and migrate to the lymphatics. One of the unique characteristics of the L3 stage is its ability to modulate and suppress the host immune response via secretion of proteins and microRNAs [6, 7].

These proteins and microRNAs allow for the initial suppression of the innate immune response which gives the opportunity for the L3 stage to mature into the adult worms

(macrofilaria). A single female macrofilaria can produce over a billion MF during this time [8, 9]. The host’s innate immune response to the MF involves many different cell populations, which include granulocytes such as eosinophils, mast cells and polymorpho- nuclear leukocytes (PMNs), as well as monocytes. These immune cell populations can recognize, trap and kill the blood circulation MF in the human filarial parasitic nematode

Brugia malayi [10, 11]. For the majority of the day, MF sequester within the lungs of the

87 host and only appear in the peripheral blood circulation for a few hours each night, which coincides with maximal mosquito feeding [12].

This parasitic nematode harbors an obligate intracellular bacteria of the Rickettsi- alaes order of α-proteobacteria called Wolbachia [13]. Wolbachia, are a vital component of larval development and adult-worm fertility and viability. Typically, Wolbachia is found within hypodermal cells of both sexes. However, vertical transmission of the bacteria to MF is facilitated by the presence of bacteria in the ovaries, oocytes, and embryos during development in females. Because of their long lifespan, mass drug treatment protocols must be maintained for between 4-5 years if the transmission is to be in- terrupted [13].

Current treatment protocols primarily focused on interrupting the spread of LF by targeting the MF larvae with combinations of anti-helminthic medications. The drugs most frequently used to manage LF include albendazole (ABZ), a benzimidazole derivative; diethylcarbamazine (DEC), a derivative of piperazine; which is involved in modulation of the host immune response to be effective [14] and ivermectin (IVM), which is believed to be a broad-spectrum antiparasitic agent [15-19]. Ivermectin has been shown in animal parasites to modulate the activity of PMNs as well as monocytes [11,

20]. These drug combinations are used primarily for long-term, community-directed treatments aimed at interrupting transmission. Some of the possible side effects of these drugs include fever, itching, and possibly oedema, arthritis, and lymphadenopathy [21].

These side effects are primarily due to the extensive paralysis and killing of the MF.

These medications have minimal activity against the causative organism, adult Wuchere- ria bancrofti, Brugia malayi and B. timori, and have thus far, failed to deliver a radical

88 curative treatment. This failure is due to the unique composition of the outer cuticle and the specificity of infected tissue limits, and the permeability of these compounds. These treatments form the foundation of the global eradication program used for LF. Current research has highlighted the use of drugs with macrofilaricidal activity [15], use of these drugs or combinations could greatly improve the probability of a curative treatment.

Attempts to develop treatments for macrofilaria have focused on the use of antimicrobials of the tetracycline class, primarily doxycycline (DOX). Use of this drug as well as the combinations of the previously mentioned anti-filarial regimens may provide adequate targeting of the symbiotic intracellular bacteria Wolbachia [17, 22]. These antimicrobials have been shown to reduce the number of resident Wolbachia, although the exact symbiotic role within the nematode is not directly known. Research has shown that the use of DOX can cause long-term sterilization and death of adult female worms in 6 to

12 months with a 3-6 week treatment course [13, 23-25]. However, limitations associated with the use of the above drugs is four-fold: specific restrictions on patient age and health status due to cytotoxicity; the inability to reach broad tissues where the adult filarial nematodes reside; as well as counter-indications in areas endemic for the Loa loa (Loi- asis) parasite; and poor patient compliance. [26]. The use of ivermectin can cause severe kill off of Loa loa MF resulting in post-ivermectin effects causing coma, irreversible neu- rologic complications and death [27]. An alternative antibiotic which has been tested and has shown effectiveness against Rickettsiae is rifampicin, which is better tolerated in children and pregnant patients and has been shown to be effective against Wolbachia in On- chocerca parasites in vitro [28]. However, rifampicin has been shown to be less effective than doxycycline in vivo [29, 30]. Additional therapeutic options for doxycycline could

89 be useful when combined with current antiparasitic drugs; however, combination therapies present difficulties for drugs that have different systemic half-lives, similar toxicity profiles, low aqueous solubility, and reduced shelf life properties. These equilibrium solubility issues can be exascebated by the differing comprehensive drug administration programs that already suffer from poor patient compliance, and multi-year therapy regimes

[31]. These issues have spurred the need for alternative drugs and rational design of delivery vehicles.

Because of the diversity of disease, location and therapies, utilization of a nanoscale delivery systems can be designed to facilitate delivery of small molecules and proteins [32-34]. Use of these delivery systems can be further enhanced through their ability to cross multiple physiological barriers readily, to targeted disease sites, all while minimizing toxicities. Most polyester nanoparticle formulations are rapidly sequestered by phagocyte cells (MPS) [32, 35-37]. Nanoparticles have also been shown to offer better pharmacokinetic properties such as controlled and sustained release, and targeting of specific tissues or organs [38]. All these features provide successful strategies for the development and utilization of amphiphilic polyanhydride nanoparticles (NP) for targeted delivery of antimicrobial drugs, against multiple diseases. Previous research has highlighted there use as a delivery vehicle for both drugs and vaccines for other diseases [15, 34, 39-

46]. Polyanhydride’s possess excellent biocompatibility and drug delivery potential, having hydrophilic properties with highly hydrophobic backbones [47]. These materials con- fer surface erosion properties that can be tailored by increasing the degree of hydrophobicity of the backbone chemistry. By tailoring the degree of hydrophobicity, polyanhydride’s can be modified to possess rapidly degrading properties (over a period of

90 hours to days) to very slow degradation (greater than a month) [48]. Such release profiles can enable single-dose therapies, whereby extending the in situ drug concentration, reduce potential drug toxicity, enhance tissue specificity and increase patient compliance.

The tailorability of the polyanhydride nanoparticles to slowly erode and release the cargo molecule in a controlled manner will increase the pharmacodynamics properties against both the adult nematode and Wolbachia.

In this work, we demonstrate the use of paired combinations of doxycycline with albendazole, diethylcarbamazine, or ivermectin through encapsulation into three different polyanhydride nanoparticle formulations, to determine a leading antiparasitic platform for efficacy testing against adult B. pahangi filarial worms. Our previous research on Brugia malayi highlighted the use of the drug combination ivermectin and doxycycline [15]. To determine the effectiveness of these drugs combined with the use of NPs against Brugia pahangi. B. pahangi is a primary parasite used in the Brugia-gerbil Meriones unguiculatus animal model. Meriones unguiculatus is used extensively for testing of drug components, and immune responses against Brugia species.

The PANs ability to penetrate the outer cuticle of the Brugia worm and effectively deliver drugs directly to the worm and its symbiotic bacteria Wolbachia at a maintained pharmacokinetic concentrations causes death. The work described herein provides insight into the use of NPs to reduce the morbidity of the filarial disease, by reducing the MF shed and improving macrofilaricidal activity.

Materials and methods

Acquisition and storage of macrofilaria and microfilariae

Live adult B. pahangi females, males, and microfilariae were acquired from the

NIH/NIAID Infectious Disease Filariasis Research Reagent Repository Center (FR3) at the University of Georgia (Athens, GA). Adult worms were maintained in non-phenol red

Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin. The macrofilaria were held in an incubator at a temperature of 37°C supplemented with 5% carbon dioxide

(CO2). Female and male worms were stored individually in 48 well microtiter plates containing 1 mL of RPMI-1640. Previously shed MF were housed in 50 mL conical tubes containing 25 mL of RPMI-1640. Upon arrival of the filarial worms, they were separated and placed into individual 48 well microtiter plates that contained the previously described media.

Motility evaluation

Ocular scoring

The motility of the adult worms treated with various formulations was observed for 30 seconds by utilizing a 2X objective on a Nikon Microscope and scored utilizing the following 0–5 scoring system: zero percent motility reduction, head and tail uninhibited as well as mid-section unaffected = 5; 1 to 25% motility reduction, ability to visualize both the head and tail movements quickly as well as midsection = 4; 26 to 49% motility reduction, showing a partial mid-section paralysis = 3; 50 to 74% motility reduction, showing full mid-section paralysis followed by a substantial reduced

92 movement in the head and tail = 2; 75 to 99% motility reduction, showing full mid- section paralysis followed by either head and or tail paralysis but not limited to occasional movement over a 30 second time period = 1; and a score of 0 represented

100% death (i.e., non-motile). The loss of motility of the adult worms was compared with that of respective untreated controls.

“Worminator” motility system

The open source computer application “WormAssay” as described by [49, 50] is a qualitative computer analysis program built to assist in large scale motility assays. The

WormAssay application (v1.4) utilized an Apple iMac computer running (v10.9.5). A

Sony (HDR-CX220) video camera (Sony, Inc.) was used as the HD video capture input for macrofilaria assays. Macroscopic, full plate assays were accomplished using a dark field illuminator and plate holder constructed similarly to the one built and described by

[49]. Worminator “Assay Analyzer” option was set to “Lucas-Kanade Optical Flow (Ve- locity, one organism per well)” with the “Plate Orientation” set to “Bottom-Read” for the

Brugia adult parasites. Filarial worms where recorded for one minute thirty seconds hourly for the first 6 hours and then daily for fifteen days thereafter. The assays were developed to have high efficiency and reproducibility and yielded objective data that is not subject to scorer bias.

Nanoparticles

For the synthesis of the CPH and CPTEG monomers, the polyanhydride polymers, 20:80 CPH:SA , 20:80 CPTEG:CPH, and 50:50 CPTEG:CPH and the ivermectin/doxycycline (IVM/DOX) and diethylcarbamazine, albendazole and doxycycline (DEC/ALB/DOX) loaded polyanhydride nanoparticles, acetic acid, acetic

93 anhydride, acetone, acetonitrile, chloroform, dimethyl formamide, ethyl ether, hexane, methylene chloride, pentane, petroleum ether, potassium carbonate, sodium hydroxide, sulfuric acid, and toluene were purchased from Fisher Scientific (Fairlawn, NJ). The chemicals 1,6-dibromohexane, 1-methyl-2-pyrrolidinone, hydroxybenzoic acid, N,N- dimethylacetamide, sebacic acid, and tri-ethylene glycol were obtained from Sigma

Aldrich (St. Louis, MO). The chemical 4-p-fluorobenzonitrile was purchased from

Apollo Scientific (Cheshire, UK). For 1H NMR analysis deuterated chloroform and deuterated dimethyl sulfoxide were purchased Cambridge Isotope Laboratories (Andover,

MA). A fluorescent dye, Rhodamine B, was purchased from Sigma Life Science (St.

Louis, MO). Ivermectin, doxycycline, diethylcarbamazine, albendazole and doxycycline were also purchased from Sigma Life Science (St. Louis, MO).

Polymer Synthesis

Synthesis of the monomers used, 1,6-bis(p-carboxyphenoxyhexane) (CPH) and

1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) was performed as described elsewhere [47]. 20:80 molar copolymers of CPTEG and CPH and CPH and SA were synthesized through melt condensation polymerization, as previously described in detail [51-53]

. Molecular weight was confirmed using 1H NMR.

Nanoparticle Fabrication

Doxycycline/ivermectin and diethylcarbamazine, albendazole and doxycycline - loaded nanoparticles of 20:80 CPH:SA , 28:80 CPTEG:CPH and 50:50 CPTEG:CPH were fabricated through solid/oil/oil nanoprecipitation, as previously described [54-56].

Drug combinations doxycycline and ivermectin, each at 5% (w/w), with Rhodamine B at

2% (w/w) were added to the polymer. Drug combinations, diethylcarbamazine, albendazole and doxycycline each at 5% (w/w), with Rhodamine B at 2% (w/w) were added to the polymer. The solid drug and polymer mixture was then dissolved in methylene chloride at a concentration of 20 mg/mL followed by rapid precipitation into the antisolvent, pentane. The polyanhydride nanoparticles were then collected using vacuum filtration.

The drug-loaded particles were characterized for size and morphology by scanning electron microscopy (SEM, FEI Quanta SEM, Hillsboro, OR).

Drug release kinetics Drug treatments of B. pahangi

Ivermectin (22,23-dihydroavermectin B1), doxycycline hyclate (C22H24N2O8

HCl 0.5H2O 0.5C2H6O), albendazole (C12H15N3O2S), diethylcarbamazine

(C10H21N3O), were dissolved in DMSO at a final concentration of 0.02% v/v. RPMI

1640 medium was prepared such that the final concentrations of each drug group tested in nanoparticles and soluble were 100, 25, 12.5, 5, 2.5, 1, .0.05, 0.05, 0.005, and 0.005

μg/ml, respectively. Control medium contained nanoparticles with 2% rhodamine B with

0.02% DMSO and rhodamine B, but no drug. Individual worms that were previously placed into 48 well flat bottom culture plates upon arrival had new fresh medium that contained 1 mL of RPMI-1640, 0.01% streptomycin and penicillin and 10% heat- inactivated fetal calf serum. B. pahangi were incubated at 37° C and 5% CO2 for 1–2 hours for acclimatization to evaluate baseline motility before the experiment began. The nanoparticle-encapsulated drugs and standard antifilarials (i.e., free drug with no nanoparticle encapsulation) were added as previously described above. For example, a dose of

25 μg of encapsulated drug contained 0.125 mg of nanoparticles w/w. Nanoparticles were added to each well, and correspondingly lower amounts of nanoparticles were added as

95 the drug concentrations decreased. Controls received equal amounts of medium but lacked the nanoparticles and standard antifilarials. Filariae were incubated at 37°C and

5% CO2 and monitored for 15 days post-treatment.

Viability assessment

Reversibility assays where conducted 24 h following a motility score of 0, throughout the treatment. Individual worms were transferred to a new 48 well microtiter plate, washed 3 times with 37° C PBS, and then 1 mL of the previously described media was added. Worms were placed in an incubator at a temperature of 37°C supplemented with 5% carbon dioxide (CO2). After 48 h an ocular and “worminator” motility score was recorded. Upon completion of reversibility adult female worm viability was assessed utilizing a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

(MTT)-formazan colorimetric assay. The same female worms used in the motility assay were gently blotted and transferred to a 96 well microtiter plate that contained 0.2 mL of

1mg/mL MTT in phosphate-buffered saline (pH 7.2) and incubated for 2 h at 37°C. The formazan formed was extracted in 0.15 mL of DMSO for 4 h at 23°C, and the absorbance was measured at 570 nm using a spectrophotometer (FLUOstar Omega version 1.01).

MF shedding and motility

The effect of treatments on MF shedding and subsequent motility was assessed using two experimental approaches. The direct effect on MF was performed by collecting

MF from the spent media of healthy untreated female worms. Spent media was centrifuged for 10 min at 200 rcf; the MF were re-suspended in fresh media and enumerated by counting using a 14.80 mm² grid plate. Aliquots containing at least 250

MF were dispensed into wells of a 96 well plate and treated in triplicate with either soluble drug, blank nanoparticles or nanoparticles containing drugs as indicated. Wells were then monitored and scored daily for MF movement. Motility of MF scoring was carried out using a 10X Nikon microscope, and individual scores from five separate fields of view (FOV) were averaged to yield a motility score for each well. Scoring was adapted from a previously reported method by observing the movements of the anterior and posterior body over one min [36]. Data were analyzed by ANOVA and graphs were assembled using GraphPad Prism (v6.05). In separate experiments, the effect of treating adult female Brugia and their ability to release MF was monitored and recorded for 14 days. The motility of the shed MF was also recorded. Each experiment, the various treatment groups, and the differing concentrations of the drugs were repeated for a minimum of three times.

Nanoparticle uptake by B. pahangi

PBS and mounted carefully onto glass slides using Prolong Gold with DAPI (Life

Technologies). Slides were then stored in the dark at 4°C before imaging. Imaging was

97 performed using an Olympus IX-71 laser-scanning confocal microscope (Fluoview 2000, v2.0) equipped with 405 nm, 488 nm, 559 nm and 635 nm lasers, spectral photomultiplier detection and a piezo-controlled stage. Laser power and exposure settings were set according to the negative (worms with no rhodamine) and positive (rhodamine containing nanoparticles) and maintained constant over all the experiments. Image stacks were collected simultaneously for bright field (transmitted light—grey scale), cell nuclei

(DAPI— blue) and nanoparticles (rhodamine—red). Bright field and cell nuclei were used to identify various tissues, organs, and structures in the worm such as oral opening, pharynx, uterus, the nerve ring, extracellular secretory apparatus, esophageal track and anal pore. Rendering of Z-stack data in three dimensions and minimal image processing was performed as described previously by our laboratory [36]using ImageJ v 1.49a

(NIH). Software parameters for image processing remained constant for all image/data sets presented.

Results

Influence of nanoparticle formulations on the fate of B. pahangi

Prior experiments assessed the benefit of NP encapsulation of IVM/DOX on B. malayi filariae revealed a benefit of co-encapsulation into nanoparticles formulated with

20:80 CPTEG:CPH chemistry. Differences in drug sensitivities for the different antiparasitic drugs, thus repeating the NP IVM/DOX in vitro experiments with B. pahangi macrofilaria to determine if a similar NP benefit exists. Female worms were incubated with decreasing amounts of drug concentration and worm motility was observed as previously described to quantify the antiparasitic activity (Figue 3-1). NP of the following polymer combinations 20:80 CPTEG:CPH, 50:50 CPTEG:CPH, and 20:80 CPH:SA were used to

98 encapsulate IVM/DOX and the fluorescent molecule rhodamine B (Rho). The only significant concentration between the nanoparticle formulations shown was the 25 μg/ml compared to the soluble. Testing of the effects of blank (i.e., no drug) and 2% Rho nanoparticles and showed no significant differences between the untreated controls.

Nanoparticle-based drug delivery increased synergism of first-line anti-filarial drug activity against B. pahangi female macrofilaria.

The effectiveness of the polyanhydride nanoparticle-based delivery platform was compared to that of the standard soluble treatment for the first line anti-filarial and antimicrobial drugs ivermectin/doxycycline. Single-drug titrations of (Figure 4-2) IVM/DOX was administered to female B. pahangi. Through ocular motility scoring the reduced motility over time was reported to average days to death (A). There were no significant differences between the nanoparticle-based delivery and soluble ivermectin/doxycycline above 25 μg/ml (not shown). The lower concentrations of NP, provided a ≤ 50% reduction in the (Fig. 3-2, A) overall time to death when compared to the soluble treated worms. The minimum effective concentrations found in the soluble group that reduced the overall days to death by <50% was observed at the 25 μg/ml concentration. Worms that did not receive either soluble or NP treatment died between 13 and 15 days.

In conjunction with the average days to death assay, females were monitored for motility with the worm assay software. A representative graph is shown at seven days post-treatment. Drug concentration responses were calculated using the worms mean motility units and percent change, compared to the untreated controls vs. drug concentration (Figure 4-2, B).

Figure 4-1 Comparison of Polyanhydride nanoparticle formulations on B. pahangi females. An average number of days to death of B. pahangi females, utilizing either soluble or encapsulated equivalent amounts of IVM/DOX and the Rho dye into three different nanoparticle formulation.Significance was determined at p<0.05, 0.01, or 0.001 as noted using a Two-Way ANOVA test. Hyper-motility was observed, where the motility of the treated parasites increased relative to the untreated controls was evident at the lower treatment concentrations in the soluble groups. The data indicated that the use of nanoparticle-based delivery significantly (p < 0.05) increased the percent inhibition when compared directly to the soluble ivermectin/doxycycline.

Reversibility and viability assays were conducted when the motility score of the worm reaches 0. Worms with a motility score of 0 were subjected to reversibility testing

100 whereby after 24 h of no motility, worms were removed from the test media, washed three times and placed in fresh media with no drugs. Reversibility of ≥ 20% paralysis was found within all soluble concentration groups. The nanoparticle-based platform had one female within the 1 μg/ml group reach a motility score of 0 and reverse, but died within

48 h. These same females were also tested for overall worm viability (as measured by the

MTT test). Testing of the viability of females (Figure 4-2, C) after 24hrs of reversibility testing, was consistent with observed average days to death and changes in motility. A representative graph of % MTT inhibition is shown for the 5 and 0.05 μg/ml drug concentrations. Inhibition of metabolic activity greater than 75% of that of untreated controls indicated that worms were non-viable and declared dead.

Soluble and nanoparticle-delivered ivermectin/doxycycline cocktail inhibit MF production in a time and concentration-dependent manner (Figure 4-2, D). Shedding in soluble treated female worms averaged ~70% of untreated at and above 5 g/ml. In contrast, NP reduced MF shedding by ~90% over the same at and above 5 μg/ml and ~70% at the lowest concentration of 0.05 ug/ml. The effect of NP on MF shedding paralleled similar activity on motility of parent female worms. The soluble ivermectin/doxycycline at concentrations above 5 μg/ml showed a relatively stable-release of MF for over the fourteen days. The minimum effective concentrations that reduced MF shedding by higher than 50% for the soluble groups was 5 μg/ml. There's the nanoparticle-based delivery system decreased overall shedding ≤ 60% at the lowest concentrations of 0.05 μg/ml.

101

NP increases antifilarial activity of diethylcarbamazine, albendazole, and doxycycline

The second-line (Figure 4-3) antifilarials drugs diethylcarbamazine and albendazole replaced ivermectin in nanoformulations and combined with doxycycline as an anti-Wolbachia agent. Following synthesis of NP with dec/abz/dox, NP and soluble drug suspensions were incubated with female and male B. pahangi worms. Results from our independent experiments for observational motility scoring, calculated average time to death (ATD), metabolic activity and MF shedding are shown in Figure 4-3. A significant (P <0.01) reduction in the average days to death when comparing the NP formulations against the soluble controls (Figure 4-3, A). NP formulations at all concentrations ranging from 25 ug/ml down to 0.05 ug/ml significantly shortened the

ATD of female worms compared to soluble controls. Soluble delivery of second-line drugs only improved killing over untreated at higher concentrations, while ≥ 1 ug/ml did not improve ATD compared to untreated. At the lowest concentration 0.05 μg/ml, the

ATD of 6.2 days for NP was half the time of both untreated controls and soluble (~13 days ATD for both). A dramatic result observed was that the lowest concentration of NP

(0.05 ug/ml) was equivalent to ATD score obtained for the highest soluble concentration of 25 ug/ml, demonstrating antiparasitic equivalent activity in NP with 100x less active drug. The soluble dec/abx/dox achieved a similar time to death, in the 5 μg/ml group (8.7 days) but required 100x more drug then the nanoparticle-based delivery. Automated worm motility scoring was also recorded for these experiments, and selected results for

5.0 and 0.05 ug/ml are shown for day 7 post-treatment in Panels B and C (Figure 4).

102

Figure 4-2 Effects of antibiotics on B. pahangi female worms. Female worms were incubated with a single concentration of soluble or encapsulated ivm/dox. (A) Average days to death (B) Percent inhibition MF motility based on Worm Assay Software. (C) Reversibility Assay-Percent Metabolic Inhibition. Dash line denoted if a tested group is considered effective if there is a 75% or greater inhibition of formazan formation compared to untreated controls. (D) Total viable MF shed by B. pahangi worms relative to control worms.

Concentration-responses were calculated using the previously mentioned parameters and compared to the untreated controls (Figure 4-3, B). Automated scoring results confirmed all significant findings that employed observational scoring. Interestingly, motility in the lower concentration 0.05 μg/ml for the soluble group consistently exceeded motility of untreated. At

103 the higher concentrations reported 5 μg/ml, there was no significance found between the two groups.

Figure 4-3 Effects of second-line antibiotics diethylcarbamazine/albendazole/doxycycline on B. pahangi female worms. Four independent experiments were conducted against Brugia female and males (not shown) worms. A total of at least five worms were incubated with a single dose titration of soluble or encapsulated DEC/ABZ/DOX. (A) Average time to death (B) Percent inhibition of motility based on Worm Assay Software. (C) Reversibility Assay-Percent Metabolic Inhibi- tion. (D) Total MF Shed by B. pahangi worms relative to control worms

104

Reversibility and viability (Figure 4-3, D) assessment was conducted as previously described. Reversibility was similar to that of the IVM/DOX groups of ≥ 27% reversibility occurred within the soluble groups. The nanoparticle-based platform had one female within the

1μg/ml group reach a motility score of 0, and upon transferring into the control media, reversed but died within 24 h. In addition to motility, worm viability was also assessed using an MTT assay (Figure 4-3, C). The nanoparticle-based delivery platform provided the most significant metabolic inhibition for the 5 and 0.05 μg/ml drug concentration. Inhibition of metabolic activity higher than 75% of that of untreated controls indicated that worms were non-viable and declared dead.

Monitoring for the total number of (i.e, viable and dead) MF shed by the female worms (Figure 4 panel D) was also conducted. The use of dec/abz/dox inhibited total MF shedd of viable Brugia females in a concentration-dependent manner. The soluble dec/abz/dox at concentrations above ≤ 5 μg/ml showed minimal effectiveness against MF shedding in Brugia females. The minimum effective concentrations that reduced MF shedding by higher than 50% for the soluble groups was 5 μg/ml. Where'as the nanoparticle-based delivery system decreased overall viable MF shed ≤ 90% at all concentrations tested.

Similar to the effects observed for adults, the average time to death of the MF treated (Figure 4-1) with the soluble drugs was significantly (p < 0.001) higher in comparison to that of the MF that were treated with the nanoparticle-based delivery system, especially at low drug concentration in the dual ivm/dox ( Figure 4-4, A) group.

The nanoparticle-based delivery system achieved a < 50% reduction in the time to death when compared to control cultures of ivermectin/doxycycline. All of the motility reductions at these concentrations are statistically significant (p < 0.01). However, the decrease

105 in time to death for the nanoparticle-based delivery of dec/abz/dox (Figure 4-4, B) the was only statistically significant (p < 0.05) at the 5 μg/ml concentration. The soluble diethylcarbamazine/albendazole/doxycycline had only minor effects on MF motility.

Confocal microscopy

To visualize the interactions of polyanhydride nanoparticles with B. pahangi female worms were treated with a cocktail of ivermectin/doxycycline with equivalent amounts rhodamine red dye either in soluble form or encapsulated within the 20:80

CPTEG:CPH nanoparticles. Detection of focal, intense rhodamine staining is consistent with the size and staining patterns of intact nanoparticles. In comparison, diffuse red staining is indicative of free rhodamine. Worms were treated for 24 h with 25 µg of the soluble drug cocktail and 20:80 CPTEG:CPH nanoparticle cocktail to determine accumulated rhodamine within tissues (Figure 4-5).

Images demonstrate the accumulation of nanoparticles within tissues throughout the worm (Figure 4-5, B) as compared to the soluble rhodamine (Figure 4-5, A) showing minimal accumulation within the body of the worm. Diffused red staining was quantified through mean intensity fluorescence (MIF) in both the soluble and nanoparticle treated worms (Figure 4-5, C). The MFI revealed the presence of rhodamine throughout the inner tissues of the worms. The total accumulated rhodamine showed increased internal staining of worms treated with 20:80 CPTEG:CPH nanoparticles. Additionally, over a period of 72 hours (Figure 4-6), a lower dose of 12.5 µg of diethylcarbamazine/albendazole/doxycycline encapsulated within the 20:80 CPTEG:CPH nanoparticles also containing the rhodamine red dye, extensively labeled the interior structures of female worms (Figure 4-6, B).

106

Figure 4-4 Effects of antibiotics on B. pahangi MF motility. MF where cultured with soluble or encapsulated antibiotics, and motility was observed over fourteen days. Results shown are average days to death.

107

Figure 4-5 Confocal microscopy of female B. pahangi with nanoparticles and soluble controls. Worms were incubated for 72 h with either soluble controls (A) or 20:80 CPTEG:CPH nanoparticles containing ivermectin (IVM), doxycycline (DOX), and rhodamine B (B) fixed and imaged by LSCM. Controls contained 25 μg each of IVM and DOX, and 0.25 μg rhodamine B, while the nanoparticles contained 25 μg of each drug and 0.25 μg of rhodamine B. Colors represent bright field image composite, DNA-Dapi (blue), rhodamine (red) and right panels are the respective bright field only. Images were collected using identical image acquisition settings. Representative images shown demonstrate the accumulation of nanoparticles within tissues throughout the worm (B) as compared to the higher amount of soluble rhodamine (A) showed minimal accumulation within the body of the worms. Mean fluorescence intensity (MFI) (C) of B. pahangi female worms shown in the relative distribution of rhodamine B comparing (A) soubly treated and (B) NP treated worms. Bar graphs represent mean ± SD (paired T-test) of relative MFI from ten equal-sized regions of interest quantified by Image J Software.

Three-dimensional reconstruction of confocal Z-stacked images shows the presence of nanoparticles and release of rhodamine within inner tissues of the worm (Figure

4-6, C). The presence of intact nanoparticles was found distributed throughout the internal reproductive organs of the worm (Figure 4-6, D).

108

Figure 4-6 Confocal microscopy of female B. pahangi with nanoparticles. (A) A schematic drawing is representing a female B. pahangi. Females range in length from 45-55 mm by 130-170 μm in width. Worms were incubated for 72 h with (B) 20:80 CPTEG:CPH nanoparticles containing DEC/ABZ/DOX, and rhodamine B, fixed and imaged by LSCM. Nanoparticles contained 12.5 μg of each drug and 0.125 of rhodamine. B Panel A is bright field image composite, rhodamine (red), DNA-Dapi (blue), and far right panel are the respective individual bright field. Inset box within the nanoparticle-treated worm outlines the area selected for side view rendering. Rep- resentative images are shown (C) demonstrate the accumulation of nanoparticles (arrows) within tissues throughout the worm. Panel D a composite image of dapi (blue) and rhodamine B (red) outlining the accumulation of nanoparticles within specific reproductive organs of the female worm. Similar results were seen throughout the body of the worm in both male and female worms (data not shown).

Discussion

The most significant barrier to the implementation of antibiotic chemotherapy for

Lymphatic filariasis has been the duration of the regimen required for permanent sterilization of female worms, or potent macrofilaricides [22]. The pathology of lymphatic filariasis is thought to be dependent on the presence of the adult worm and

109 interruption can bring direct relief to individual patients. The use of antifilarials such as

Diethylcarbamazine is primarily useful as a microfilaricide and partially effective macrofilaricide. Ivermectin and albendazole are broad-spectrum anthelmintics that have been shown to decrease the prevalence through interruption and transmission of MF [57].

Currently, the use of doxycycline or tetracyclines as a safe and effective drug for adult macrofilaria sterilization has been shown to be effective at targeting the symbiont

Wolbachia. The incorporation of doxycycline has also been shown to have long-term embryostatic effects [24, 58, 59]. However, previously published doxycycline regimens are lengthy, and a shorter regimen is desirable to complement classical anti-filarial drugs.

Such studies have highlighted the use of other drugs active against rickettsia-like organisms such as rifampicin. These studies have found that short course, high concentration therapies provide comparable depletion of the symbiont Wolbachia thereby reducing the embryogenic capacity within macrofilariae [59]. However, Rifampicin is a known potent inhibitor of cytochrome P450 enzymes (CYPs) which are clinically relevant in the metabolism and elimination of drugs.

The present studies have confirmed our previous research that focused on the effects of encapsulated antibacterial antibiotics on Brugia malayi and extended our understanding of this topic in several ways [15]. In this study, we report the use of a polyanhydride nanoparticle-based carrier to efficiently reduce the amount of drug necessary for macrofilariae death by up to 200-fold compared to physiologically relevant doses. The three polymer combinations (Figure 4-1) 20:80 CPTEG:CPH, 50:50 CPTEG:CPH, and

20:80 CPH:SA each containing encapsulated Ivermectin and Doxycycline at various concentrations. The use of a nanoparticle only formulation(i.e., no drug) and found

110 minimal activity against Brugia. The nanoparticle formulations (Figure 4-1) were relatively similar in their effectiveness in the delivering of drugs to Brugia adults. There were slight differences in the higher concentrations tested, this small decrease in killing time for the 20:80 CPH:SA group is due to the increased amounts of SA and decreased relative quantities CPH content. This polymer combination results in a more rapid release of the encapsulated drug, which is consistent with previous work. The 20:80

CPTEG:CPH group provided the most systematic killing between the formulations.

Previous work has shown that this copolymer composition contains weakly segregated

CPTEG- and CPH-rich domains [60]. This composition may indicate a preferential partitioning of the hydrophobic ivermectin to the slowly degrading CPH-rich domains of the copolymer; this may explain the slight differences between the killing time of the nanoparticles tested.

Both soluble ivermectin/doxycycline and diethylcarbamazine/albendazole/doxycycline (Figure 4-2) provided a minimal macrofilaricidal activity for the concentration ranges tested. The polyanhydride nanoparticle drug delivery platform decreased the average time to death of B. pahangi females and facilitated a reduction in the total number of the MF shed as shown in Figure 4-2. Rapid killing would likely contribute to the dramatic reduction in shedding by female worms at low nanoparticle concentrations (Figure 4-2, C and D). The use of diethylcarbamazine/albendazole/doxycycline was most effective as a microfilaricide (Figure 4-4). It is also likely that the nanoparticles increased the rate of delivery of ivermectin and rapidly paralyzed the muscles within the body of the worm as well as the vulva, which is needed to expel the MF. Similar trends were also observed in the MF to a slightly lesser extent in the

111 ivermectin/doxycycline groups possibly reflecting the comparatively lower number of

Wolbachia in MF compared to female macrofilariae (Figure 4-4).The diethylcarbamazine/albendazole/doxycycline nanoparticle groups a significant reduction in viability of the

MF over the course of treatment.

As discussed above, the encapsulation and subsequent controlled release of the drug and the ability of the polyanhydride nanoparticle carriers to localize to the worm cuticle surface, and even be internalized by the worms, lead to a higher microenvironment concentration of the drugs. In contrast, the soluble drug cocktail is in an aqueous form, and so to create the same concentrations internally to the worm, more drug must be administered initially to cross the complex cuticle surface. This is supported by the confocal microscopy data in Figure 4-5 and 4-6, which show that the polyanhydride nanoparticles readily localize to the worm, increasing the concentration of the internalized dye as compared to the soluble control. The confocal microscopy data in

Figure 4-6 provides an initial observation of the strong interactions between polyanhydride nanoparticles and the cuticle surface, which is hypothesized to lead to the efficacy profile of this nano-carrier platform. Tracking (Figure 4-5, C) the fluorescent rhodamine

B co-loaded into the nanoparticles demonstrated a more significant increased influx of payload into the worm compared to drug and dye delivered solubly. In addition to providing sustained release of the drug, the drug-loaded polyanhydride nanoparticles demonstrated enhanced internalization by Brugia worms. The enhanced internalization properties are evident based on the exposure amount and total retained dye. We speculate that the nanoparticles undergo facilitated diffusion into the worm, by crossing the cuticle,

112 penetrating the hypodermis membrane barriers and accessing deeper tissues within the worm [61, 62].

This data taken together provides us the necessary tools and understanding to incorporate the use of a nanoparticle-based delivery platform. This platform can efficiently target the symbiont Wolbachia through use of encapsulated antimicrobials thereby interrupting embryogenesis. It is also evident that utilization of this type of delivery system can directly target and eliminate the macrofilaria. This can be achieved through interrupting the spread of disease and reducing the drug concentrations necessary to eliminate and effectively clearing the disease.

References

1. Grote, A., et al., Defining Brugia malayi and Wolbachia symbiosis by stage- specific dual RNA-seq. PLoS Negl Trop Dis, 2017. 11(3): p. e0005357.

2. Disease, G.B.D., I. Injury, and C. Prevalence, Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet, 2017. 390(10100): p. 1211-1259.

3. Organization, W.H. Global tuberculosis report 2017. 2017 [cited 2017 December 01]; Available from: http://www.who.int/tb/publications/global_report/en/.

4. O'Neill, M., et al., Potential Role for Flubendazole in Limiting Filariasis Transmission: Observations of Microfilarial Sensitivity. Am J Trop Med Hyg, 2017.

5. Control, C.f.D. Parasites - Lymphatic Filariasis. 2010 June 14, 2013 [cited 2017 December 11]; Available from: https://www.cdc.gov/parasites/lymphaticfilariasis/biology.html.

6. Buck, A.H., et al., Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity. Nat Commun, 2014. 5: p. 5488.

113

7. McSorley, H.J., J.P. Hewitson, and R.M. Maizels, Immunomodulation by helminth parasites: defining mechanisms and mediators. Int J Parasitol, 2013. 43(3-4): p. 301-10.

8. Bandi, C., et al., Phylogeny of Wolbachia in filarial nematodes. Proc Biol Sci, 1998. 265(1413): p. 2407-13.

9. Hoerauf, A., et al., Filariasis in Africa--treatment challenges and prospects. Clin Microbiol Infect, 2011. 17(7): p. 977-85.

10. Greene, B.M., H.R. Taylor, and M. Aikawa, Cellular killing of microfilariae of Onchocerca volvulus: eosinophil and neutrophil-mediated immune serum- dependent destruction. J Immunol, 1981. 127(4): p. 1611-8.

11. McCoy, C.J., et al., Human Leukocytes Kill Brugia malayi Microfilariae Independently of DNA-Based Extracellular Trap Release. PLoS Negl Trop Dis, 2017. 11(1): p. e0005279.

12. Schroeder, J.H., et al., Live Brugia malayi microfilariae inhibit transendothelial migration of neutrophils and monocytes. PLoS Negl Trop Dis, 2012. 6(11): p. e1914.

13. Hoerauf, A., et al., Endosymbiotic bacteria in worms as targets for a novel chemotherapy in filariasis. Lancet, 2000. 355(9211): p. 1242-3.

14. Maizels, R.M. and D.A. Denham, Diethylcarbamazine (DEC): immunopharmacological interactions of an anti-filarial drug. Parasitology, 1992. 105 Suppl: p. S49-60.

15. Binnebose, A.M., et al., Polyanhydride Nanoparticle Delivery Platform Dramatically Enhances Killing of Filarial Worms. PLoS Negl Trop Dis, 2015. 9(10): p. e0004173.

16. Molyneux, D.H., et al., Mass drug treatment for lymphatic filariasis and onchocerciasis. Trends Parasitol, 2003. 19(11): p. 516-22.

17. Supali, T., et al., Doxycycline treatment of Brugia malayi-infected persons reduces microfilaremia and adverse reactions after diethylcarbamazine and albendazole treatment. Clin Infect Dis, 2008. 46(9): p. 1385-93.

18. Talbot, J.T., et al., Predictors of compliance in mass drug administration for the treatment and prevention of lymphatic filariasis in Leogane, Haiti. Am J Trop Med Hyg, 2008. 78(2): p. 283-8.

19. Al-Abd, N.M., et al., Antifilarial and Antibiotic Activities of Methanolic Extracts of Melaleuca cajuputi Flowers. Korean J Parasitol, 2016. 54(3): p. 273-80.

114

20. Vatta, A.F., et al., Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro. Vet Parasitol, 2014. 206(1-2): p. 38-42.

21. Poolman, E.M. and A.P. Galvani, Modeling targeted ivermectin treatment for controlling river blindness. Am J Trop Med Hyg, 2006. 75(5): p. 921-7.

22. Taylor, M.J., et al., Anti-Wolbachia drug discovery and development: safe macrofilaricides for onchocerciasis and lymphatic filariasis. Parasitology, 2014. 141(1): p. 119-27.

23. Sungpradit, S., T. Chatsuwan, and S. Nuchprayoon, Susceptibility of Wolbachia, an endosymbiont of Brugia malayi microfilariae, to doxycycline determined by quantitative PCR assay. Southeast Asian J Trop Med Public Health, 2012. 43(4): p. 841-50.

24. Turner, J.D., et al., Macrofilaricidal activity after doxycycline only treatment of Onchocerca volvulus in an area of Loa loa co-endemicity: a randomized controlled trial. PLoS Negl Trop Dis, 2010. 4(4): p. e660.

25. Taylor, M.J., et al., Macrofilaricidal activity after doxycycline treatment of Wuchereria bancrofti: a double-blind, randomised placebo-controlled trial. Lancet, 2005. 365(9477): p. 2116-21.

26. Njambe Priso, G.D., et al., Filaria specific antibody response profiling in plasma from anti-retroviral naïve Loa loa microfilaraemic HIV-1 infected people. BMC Infectious Diseases, 2018. 18(1): p. 160.

27. Drame, P.M., et al., Identification and Validation of Loa loa Microfilaria-Specific Biomarkers: a Rational Design Approach Using Proteomics and Novel Immunoassays. mBio, 2016. 7(1): p. e02132-15.

28. Townson, S., et al., Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro. Filaria J, 2006. 5: p. 4.

29. Hoerauf, A., et al., Efficacy of 5-week doxycycline treatment on adult Onchocerca volvulus. Parasitol Res, 2009. 104(2): p. 437-47.

30. Specht, S., et al., Efficacy of 2- and 4-week rifampicin treatment on the Wolbachia of Onchocerca volvulus. Parasitol Res, 2008. 103(6): p. 1303-9.

31. Tamarozzi, F., et al., Long term impact of large scale community-directed delivery of doxycycline for the treatment of onchocerciasis. Parasit Vectors, 2012. 5: p. 53.

115

32. Walkey, C.D., et al., Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake. J Am Chem Soc, 2012. 134(4): p. 2139-47.

33. del Pozo, J.L. and R. Patel, The challenge of treating biofilm-associated bacterial infections. Clin Pharmacol Ther, 2007. 82(2): p. 204-9.

34. Ross, K.A., et al., Nano-enabled delivery of diverse payloads across complex biological barriers. J Control Release, 2015. 219: p. 548-559.

35. Wafa, E.I., et al., The effect of polyanhydride chemistry in particle-based cancer vaccines on the magnitude of the anti-tumor immune response. Acta Biomater, 2017. 50: p. 417-427.

36. Ulery, B.D., et al., Polymer chemistry influences monocytic uptake of polyanhydride nanospheres. Pharm Res, 2009. 26(3): p. 683-90.

37. Phanse, Y., et al., Analyzing cellular internalization of nanoparticles and bacteria by multi-spectral imaging flow cytometry. J Vis Exp, 2012(64): p. e3884.

38. Malam, Y., E.J. Lim, and A.M. Seifalian, Current trends in the application of nanoparticles in drug delivery. Curr Med Chem, 2011. 18(7): p. 1067-78.

39. Berkland, C., et al., Microsphere size, precipitation kinetics and drug distribution control drug release from biodegradable polyanhydride microspheres. J Control Release, 2004. 94(1): p. 129-41.

40. Brenza, T.M., et al., Neuronal protection against oxidative insult by polyanhydride nanoparticle-based mitochondria-targeted antioxidant therapy. Nanomedicine, 2017. 13(3): p. 809-820.

41. Brenza, T.M., et al., Pulmonary biodistribution and cellular uptake of intranasally administered monodisperse particles. Pharm Res, 2015. 32(4): p. 1368-82.

42. Carrillo-Conde, B., et al., Protein adsorption on biodegradable polyanhydride microparticles. J Biomed Mater Res A, 2010. 95(1): p. 40-8.

43. Carrillo-Conde, B., et al., Encapsulation into amphiphilic polyanhydride microparticles stabilizes Yersinia pestis antigens. Acta Biomater, 2010. 6(8): p. 3110-9.

44. Chavez-Santoscoy, A.V., et al., Tailoring the immune response by targeting C- type lectin receptors on alveolar macrophages using "pathogen-like" amphiphilic polyanhydride nanoparticles. Biomaterials, 2012. 33(18): p. 4762-72.

116

45. Narasimhan, B., J.T. Goodman, and J.E. Vela Ramirez, Rational Design of Targeted Next-Generation Carriers for Drug and Vaccine Delivery. Annu Rev Biomed Eng, 2016. 18: p. 25-49.

46. Vela-Ramirez, J.E., et al., Safety and biocompatibility of carbohydrate- functionalized polyanhydride nanoparticles. AAPS J, 2015. 17(1): p. 256-67.

47. Torres, M.P., et al., Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. J Biomed Mater Res A, 2006. 76(1): p. 102-10.

48. Lopac, S.K., et al., Effect of polymer chemistry and fabrication method on protein release and stability from polyanhydride microspheres. J Biomed Mater Res B Appl Biomater, 2009. 91(2): p. 938-47.

49. Marcellino, C., et al., WormAssay: A Novel Computer Application for Whole- Plate Motion-based Screening of Macroscopic Parasites. PLoS Neglected Tropical Diseases, 2012. 6(1): p. e1494.

50. Storey, B., et al., Utilization of computer processed high definition video imaging for measuring motility of microscopic nematode stages on a quantitative scale: “The Worminator”. International Journal for Parasitology: Drugs and Drug Resistance, 2014. 4(3): p. 233-243.

51. Domb, A.J., et al. Polyanhydrides: Synthesis and characterization. 1993. Berlin, Heidelberg: Springer Berlin Heidelberg.

52. Kipper, M.J., et al., Design of an injectable system based on bioerodible polyanhydride microspheres for sustained drug delivery. Biomaterials, 2002. 23(22): p. 4405-12.

53. Torres, M.P., et al., Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. Journal of biomedical materials research. Part A, 2006. 76(1): p. 102.

54. Haughney, S.L., et al., Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles. Acta Biomater, 2013. 9(9): p. 8262-71.

55. Ulery, B.D., et al., Design of a protective single-dose intranasal nanoparticle- based vaccine platform for respiratory infectious diseases. PLoS One, 2011. 6(3): p. e17642.

56. Huntimer, L., et al., Evaluation of biocompatibility and administration site reactogenicity of polyanhydride-particle-based platform for vaccine delivery. Adv Healthc Mater, 2013. 2(2): p. 369-78.

117

57. Ottesen, E.A., et al., The Global Programme to Eliminate Lymphatic Filariasis: Health Impact after 8 Years. PLoS Neglected Tropical Diseases, 2008. 2(10): p. e317.

58. Landmann, F., et al., Anti-filarial activity of antibiotic therapy is due to extensive apoptosis after Wolbachia depletion from filarial nematodes. PLoS Pathog, 2011. 7(11): p. e1002351.

59. Turner, J.D., et al., Albendazole and antibiotics synergize to deliver short-course anti-Wolbachia curative treatments in preclinical models of filariasis. Proc Natl Acad Sci U S A, 2017. 114(45): p. E9712-E9721.

60. Shen, E., et al., Microphase separation in bioerodible copolymers for drug delivery. Biomaterials, 2001. 22(3): p. 201-10.

61. Howells, R.E., A.M. Mendis, and P.G. Bray, The mechanisms of amino acid uptake by Brugia pahangi in vitro. Z Parasitenkd, 1983. 69(2): p. 247-53.

62. Luck, A.N., et al., Heme acquisition in the parasitic filarial nematode Brugia malayi. The FASEB Journal, 2016. 30(10): p. 3501-3514.

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CHAPTER 5 . THE EFFECTIVENESS OF A NANO-BASED DELIVERY PLATFORM AGAINST MYCOBACTERIUM MARINUM

Manuscript to be submitted to Antimicrobial Agents and Chemotherapy

Andrea M. Binnebose1, Adam S. Mullis 2, Shannon L. Haughney2, Balaji Narasimhan2*, and Bryan H. Bellaire1*

1 Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, 50011 2 Department of Chemical and Biological Engineering, Iowa State University, Ames, 50011

* To whom all correspondence must be addressed

E-mail: [email protected]; Phone: (262) 384-1086, [email protected]; Phone: (515) 294-1006, [email protected]; Phone:(515) 294-8019

Abstract

Mycobacterium marinum is a waterborne pathogen responsible for tuberculosis- like infections in ectotherms and is an occasional opportunistic human pathogen. M. marinum is the closest genetic relative of the M. tuberculosis complex and as such is the most reliable in testing for drug susceptibility. The use of a multi-drug therapy has become the standard treatment for tuberculosis. The Mycobacterium species show a high degree of inherent resistance to most antibiotics and chemotherapeutic agents due to their unique cellular membrane. The cell walls high lipid content, and unusual structure forms an impenetrable barrier that inhibits the entry of hydrophilic/hydrophobic antitubercular drugs. Herein, we describe a method utilizing amphiphilic polyanhydride nanoparticles as a drug delivery system against Mycobacterium, in developing a new drug delivery platform that can overcome the multi-facilitated inherent resistant nature of the bacteria.

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Results showed that the use of the polyanhydride nanoparticle increased internalized by macrophages, efficiently trafficked to lysosomes, and release antituberculosis drugs intracellularly. Our results are consistent with nanoparticles improving the elimination of intracellular Mycobacteria by increasing intracellular targetting of the antimicrobial drug cocktail. Combined with previously published results illustrating the capacity of NP to enhance soluble delivery of antibiotics against intracellular pathogens,, these data support the use of polyanhydride nanoparticles as a versatile platform that can dramatically improve intracellular targetting with potential use as a treatment against Mycobacteria infections.

Introduction

The world health organization (WHO) has reported that in 2016, 6.3 million new cases of Mycobacterium tuberculosis were reported and nearly 1.3 million individuals died as a result [1]. M. tuberculosis is the ninth leading cause of death worldwide and the leading cause of death from a single infectious agent [1, 2]. Of these individuals infected almost 10% of the new cases reported were found to be resistance to the first line drugs, rifampicin and an additional 5% of new cases reported multi-drug resistance (MDR-TB) strains. These strains are reported to be resistant to at least rifampicin (RIF) and isoniazid

(INH) [3-5]. Individuals that succumb to this disease could be prevented with appropriate treatment and diagnosis. The clinical features of tuberculosis (TB) infection include multistage processes of primary infection, latency, and reactivation. Years of efforts have been made to reduce the adverse treatment and develop an understanding of the pathogenesis of the organism [6-8]. Researchers have used multiple mycobacteria species to study the pathogenesis and resistance mechanisms of tuberculosis, including M.

120 smegmatis, M. bovis, and M. marinum. Of these models, the nontuberculous M. marinum is the closest genetic relative of the M. tuberculosis complex [9] and causes TB-like granuloma formation in ectotherms [10]. Eighty-five percent of M. marinum loci encoding recognized virulence genes have homologous genes in M. tuberculosis [11]. Thus, due to its similar pathogenicity, granuloma formation, as well as latency profile M. marinum, is widely used as a model to study infections, and drug screening has been evaluated [11-

14].

Nontuberculous mycobacteria (NTM) are responsible for a multiplicity of different types of infections, including respiratory, cutaneous, and systemic [15], of which many of the species of NTM are multidrug-resistant [16], including M. marinum. M. marinum is an atypical bacilli’s that are reported to be resistant to the anti-tuberculosis medications isoniazid, pyrazinamide, and para-aminosalicylic acid and shows intermediate sensitivity to streptomycin [17, 18]. Unlike M. tuberculosis slow growth rate of 3-6 weeks, M. marinum grows within 1-2 weeks. This faster growth rate allows for a model that can be correlated to effective drug susceptibility in vitro.

Another hurdle in the testing of Mycobacterium is there complex cellular wall

[19, 20] and the use of antibiotics with a hydrophilic/hydrophobic nature [21, 22], as well as the use of prodrugs (metabolically activated) in the treatment of TB [23, 24]. Most soluble free drugs have been found to be internalized within macrophages [25, 26] and stored within the lysosomes, where the bioactivity of the drug is low [27]. Mycobacte- rium’s ability to prevent the enzymatic conversion of these prodrug antibiotics [28] interferes with the capacity to act efficiently upon the bacteria due to their slow growth rate

[29] and failure to co-localize efficiently within Mycobacteria containing endosomes.

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Therefore, limited intracellular activity against non-replicating bacteria is often found.

Because the therapeutic efficacy of most drugs has been well recognized, inefficient delivery could result in an insufficient therapeutic index, causing persistent and latent infection.

One approach to improving the efficacy of single drug therapy has been to design treatment regimens based on the pharmacodynamics of antibiotics over time. Current treatment for M. marinum infections includes the use of the antibiotics streptomycin and gentamicin, with doxycycline as an emerging alternative therapy. Although fluoroquinolones such as ciprofloxacin have demonstrated success in research settings, these drugs are not widely used on humans. Additionally, treatment must be administered rapidly after the onset of symptoms for optimal efficacy [30].

Previous research has demonstrated the increased drug efficacy, distribution, and sustained antiparasitic effects of co-encapsulating the antimicrobials doxycycline along with multiple antifilarials overextend time points (i.e., up to 14 days) with polyanhydride nanoparticles [31]. These nanoparticles were based on 1,6-bis(pcarboxyphenoxy)hexane

(CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG). With the use of these polymer and copolymer combinations, synergism was observed between drug and encapsulation into nanoparticles resulting in increased antimicrobial activity through the sustained release of sub-inhibitory concentrations. Varying polymer and copolymer chemistry combinations of the nanoparticle contributed to the antimicrobial activity of different drug combinations. In this work, the effect of nanoparticle chemistry was analyzed against the intracellular pathogen M. marinum. The use of the agar dilution method is

122 considered the most accurate reference method for Mycobacterium marinum drug susceptibility testing [32]. The development, effectiveness and increased susceptibility of a polyanhydride nanoparticle delivery system were explored using the first line antitubercular drugs rifampicin, isoniazid, pyrazinamide and ethambutol against M. marinum.

Materials and methods

Nanoparticles

For the synthesis of the CPH and CPTEG monomers, the polyanhydride polymers, 20:80 CPH:SA , 20:80 CPTEG:CPH, and 50:50 CPTEG:CPH and the rifampicin, isoniazid, pyrazinamide and ethambutol-loaded polyanhydride nanoparticles, acetic acid, acetic anhydride, acetone, acetonitrile, chloroform, dimethyl formamide, ethyl ether, hexane, methylene chloride, pentane, petroleum ether, potassium carbonate, sodium hydroxide, sulfuric acid, and toluene were purchased from Fisher Scientific

(Fairlawn, NJ). The chemicals 1,6-dibromohexane, 1-methyl-2-pyrrolidinone, hydroxybenzoic acid, N,N-dimethylacetamide, sebacic acid, and tri-ethylene glycol were obtained from Sigma Aldrich (St. Louis, MO). The chemical 4-p-fluorobenzonitrile was purchased from Apollo Scientific (Cheshire, UK). For 1H NMR analysis deuterated chloroform and deuterated dimethyl sulfoxide were purchased Cambridge Isotope Laborato- ries (Andover, MA). A fluorescent dye, Rhodamine B, was purchased from Sigma Life

Science (St. Louis, MO). Rifampicin, isoniazid, pyrazinamide, and ethambutol were also purchased from Sigma Life Science (St. Louis, MO).

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Polymer Synthesis

Synthesis of the monomers used, 1,6-bis(p-carboxyphenoxyhexane) (CPH) and

1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) was performed as described elsewhere [33]. 20:80 molar copolymers of CPTEG and CPH and CPH and SA were synthesized through melt condensation polymerization, as previously described in detail [34-36]

. The molecular weight was confirmed using 1H NMR.

Nanoparticle Fabrication

Rifampicin, isoniazid, pyrazinamide, and ethambutol -loaded nanoparticles of 20:80

CPH:SA and 28:80 CPTEG:CPH were fabricated through solid/oil/oil nanoprecipitation, as previously described [37-39]. Drug combinations Rifampicin and pyrazinamide, each at 5% (w/w), with Rhodamine B at 2% (w/w) were added to the polymer. Drug combinations, isoniazid and ethambutol each at 5% (w/w), with Rhodamine B at 2% (w/w) were added to the polymer. The solid drug and polymer mixture was then dissolved in methylene chloride at a concentration of 20 mg/mL followed by rapid precipitation into the antisolvent, pentane. The polyanhydride nanoparticles were then collected using vacuum filtration. The drug-loaded particles were characterized for size and morphology by scanning electron microscopy (SEM, FEI Quanta SEM, Hillsboro, OR).

Bacterial strains and growth conditions

M.marinum (ATCC 927) was propagated at 30°C either in Middlebrook 7H9 broth (Difco) containing 10% oleic acid-albumin-dextrose-catalase (Difco) and 0.05%

Tween 80 (Sigma) or on Middlebrook 7H10 agar containing 10% oleic acid-albumin- dextrose-catalase. Frozen stocks were prepared by growing bacteria in 7H9 broth to mid-

124 log phase (optical density at 600 nm [OD600], ∼0.5), collecting cells by centrifugation, washing once with phosphate-buffered saline containing 0.05% Tween 80 (PBST), adding glycerol to 15%, and storing in aliquots at −80°C. Bacterial stocks were titered by plating PBST-diluted bacterial suspensions on 7H10 agar and scoring colonies after 1 week at 30°C.The recombinant strain of M. marinum expressing the green fluorescent protein (M. marinum- GFP) bears an integrative plasmid (pGFPHYG2). The pGFPHYG2 was a gift from Lalita Ramakrishnan (Addgene plasmid # 30173), and electroporation and selection of transformants were carried out as previously described [40].

Drug solutions were prepared at concentrations of 20 mg/ml (PZA; Sigma-Al- drich) and 2 mg/ml in distilled water (INH, and EMB; Sigma-Aldrich) or dimethyl sulfoxide (RIF; Sigma-Aldrich), filter sterilized and frozen until used.

Cells and culture conditions

The human monocytic cell line THP-1 was cultured in RPMI 1640 medium 10%

(v/v) heat-inactivated fetal bovine serum. Suspension cultures of THP-1 cells were maintained at a cell density between 5 x 105 and 1 x 106 cells/ml of cell culture medium and split twice per week. THP-1 cells growing in suspension were harvested at a density of 1

× 106 cells/ml, resuspended in fresh medium supplemented with 5 nM phorbol myristic acid (PMA) to differentiate the cells into adherent monocytes, and placed into 96 well tissue culture plates or 24-well tissue (Costar) culture plate containing sterile #1 glass coverslips in each well. After overnight culture in the presence of PMA, adherent cells were washed three times gently with phosphate-buffered saline (PBS; pH 7.4) and incubated for an additional 24 h in complete RPMI 1640 medium with no PMA.

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Broth microdilution method

Antibiotic testing against M. marinum was determined in 7H9 broth by the standard microdilution method. Briefly, 0.1-ml of a 0.1 mg ml-1 dilution of previously mentioned drugs were added to a 96-well plate. Control wells included medium and culture controls. One hundred microliters of culture (at 0.5 x 106 CFU/ml) was added to all the wells except the medium control wells. The plates were incubated at 30°C for up to 72 h.

Following each incubation time intervals, twenty microlitres of each bacterial suspension were serially diluted in PBS and aliquots of triplicate were plated onto Middlebrook

7H10 or 7H11 media. The plates were sealed and incubated for 1-2 weeks at 30°C. Ex- periments were conducted in triplicate and the standard deviation is depicted on graphs unless otherwise stated.

Intracellular survival of Mycobacterium

THP-1 cells used for evaluation of the intracellular survival of M. marinum were treated similarly as previously described in 96-well flat-bottom tissue culture plates. Bac- terial suspensions were prepared and generated by scraping 14 d cultures of the M. marinum grown on Middlebrook 7H10 agar into screw-cap microfuge tubes containing 7H9 media and incubated for 96 h. Pellets of bacteria were resuspended through mechanical disruption and numbers of bacteria present in the suspensions were determined by optical density at 600 nm measurements[41]. These suspensions were used to generate dilute mycobacteria using complete RPMI 1640 medium whereby the bacterial density was adjusted to desired levels to account for variations in the numbers of target monocytes. Sus- pensions of bacteria were added to monocyte monolayers at a multiplicity of infection

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(bacteria/monocyte ratio) of 1:1 and 10:1. Tissue culture plates were gently agitated by hand and then centrifuged at 4°C for 10 min at 270 x g. Moreover, transferred to an incubator for 90 min 37°C with 5% CO2 to allow for phagocytosis of bacteria. Monolayers were washed three times with PBS to remove any remaining nonadherent bacteria. Fresh

RPMI-1640 with 10 ug/ml gentamicin complete media was added following the last washing. The viability of intracellular Mycobacterium was determined by lysing monocytes with 0.1% deoxycholate, diluting suspensions in PBS, and plating aliquots in triplicate on Middlebrook 7H10 medium. The plates were sealed and incubated for up to 14 days at 30°C. Total cfu/ml of bacterial survival at 24, 48 and 72 h were calculated based on the number of internalized bacteria detected at 2 h postinfection which represents

100% of internalized bacteria. All minimum inhibitory concentration testing and presentation was conducted under the Clinical and Laboratory Standards Institute (CLSI) rec- ommendations (CLSI., 2015). Experiments were performed in triplicate and the standard deviation is depicted on graphs unless otherwise stated

Immunofluorescence microscopy.

Coverslips harboring the adherent and infected monocytes were fixed with freshly prepared 4% paraformaldehyde in PBS (pH 7.4) at room temperature for a minimum of

20 min. Following fixation, differential staining was initiated by washing monolayers with cold PBS, followed by incubating coverslips in PBS at 1 h at 4°C successively with mouse Lamp-1 antibody (1/200), followed with IgG-specific anti-rabbit secondary antibodies conjugated with Alexa 647 fluorochromes (1/200) [42]. Monolayers were routinely permeabilized with bovine serum albumin-PBS solution containing a final concentration of 0.1% saponin (BSP). Incubations with primary and secondary antibodies (at

127 concentrations indicated above) were at room temperature 4 h, at which time coverslips were washed three times with cold BSP and affixed to a glass slide with Pro-long mount- ing solutions. Immunofluorescence microscopy was performed using an Olympus IX-81 laser-scanning confocal microscope (Fluoview 2014, v4.2) equipped with 405 nm, 488 nm, 559 nm and 635 nm lasers, spectral photomultiplier detection and a piezo-controlled stage. Inverted Olympus IX-71 fluorescence microscope equipped with environment control chamber, vibration table. 4 filter sets for imaging DAPI, GFP/FITC and

TRITC/Texas Red equipped with zenon Arc lamp, Nipkow-Confocal Module, high resolution-Hamamatsu CCD, and Metamorph Imaging Suite for microscope automation and image capture/analysis software (v. 4.12) . Fields of view were imaged using 40x apochromatic objective, and all image exposure conditions were maintained throughout the experiment. Minimal image processing was performed as described previously by our laboratory using ImageJ v 1.51n (NIH) [31]. Software parameters for image processing remained constant for all image/data sets presented.

Results

Antimicrobial activity of single-drugs in broth cultures To assess the benefits of encapsulating antitubercular drugs into NP, we began by examining the antimicrobial activity of first-line drugs individually against broth cultures of M. marinum. Early log phase M. marinum cultures were exposed to rifampin, pyrazinamide, isoniazid and ethambutol in a single soluble concentration or encapsulated into nanoparticles. Following 48 hours of incubation, aliquots of cultures were serially diluted and plated on a solid agar medium to enumerate viable bacteria. Individual drug sensitivity results for single drugs tested against M. marinum are presented in detail in Figure 5-1.

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Bacterial counts from untreated were used to set the limit representing inhibition of 90% of M. marinum (MIC90). The calculated MIC90 concentrations for the 4 soluble drugs fell within expected ranges for rifampicin at 2 μg/ml, ethambutol at 20 μg/ml, isoniazid at 5 μg/ml and greater than < 32μg of pyrazinamide. M. marinum is inherently resistant to pyrazinamide due to poor membrane diffusion owed to unique membrane permeability for this species. NP formulations containing rifampicin with 20:80 CPTEG:CPH chemistry reduced the MIC90 to 0.09 μg/ml, and

20:80 CPH:SA (1.25 μg/ml). MICs for isoniazid in the 20:80 CPTEG:CHA group (5 μg/ml),

MIC50 for ethambutol and pyrazinamide (20 μg/ml). MICs for 20:80 CPH:SA for ethambutol, isoniazid, and pyrazinamide all exceeded CSLI breakpoints. There appears to be a linear growth trend that is neither drug nor concentration-dependent.

The effectiveness of multi-tubercular drugs

Results from the single drug assay provided the necessary information for con- struction of a dual-encapsulated nanoparticle Figure 5-2. Encapsulation of first-line drugs rifampicin and pyrazinamide, into either 20:80 CPTEG:CPH or 20:80 CPH:SA. A single concentration of either soluble and or encapsulated rifampicin/pyrazinamide in nanoparticles appears to improve the pyrazinamide’s mode of action against M. marinum. Use of

NP with encapsulated rifampicin/pyrazinamide reduced the bacterial concentration 1.45 logs CFU (± 0.05) when compared to the single PZA drug. This 10 % reduction in viable bacteria was found at the 0.05μg/ml concentration for (A) 20:80 CPTEG:CPH. The 20:80

CPH”SA NP (C) provided a 1.59 log CFU (± 0.10) reduction. This is in stark contrast to the soluble groups.

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Figure 5-1 M. marinum drug sensitivity profiles for rifampicin and other antibiotics in broth culture model. Drug susceptibility of M. marinum in broth culture media after 48 h exposure to (A) rifampicin, (B) pyrazinamide, (C) isoniazid, and (D) ethambutol as a single treatment dose of soluble or encapsulated in 20:80 CPTEG:CPH nanoparticle. All data are represented as Colony forming units/ml (CFU).

Interestingly, the addition of rifampicin with pyrazinamide reduced the antimicrobial activity measured for rifampicin alone, suggesting an antagonistic relationship between the administration of these drugs. This reduction in antimicrobial activity was observed in both soluble and NP delivered groups while less dramatic for the 20:80

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CPTEG:CPH formulation improve upon rifampicin alone in NP. In contrast, the combination of ethambutol with isoniazid improved bacterial killing at higher concentrations and NP treated cultures had significantly lower CFU at both 32 µg/ml and 8 µg/ml. Inef- fectiveness of ethambutol was improved with the addition of isoniazid when used in the

(B) 20:80 CPTEG:CPH nanoparticle. The concentration of 0.125 μg/ ml a 1.28 log CFU

(± 0.29) reduction in bacteria which resulted in a 9 % decrease of viable bacteria compared to the single drugs. The continued trend for either 20:80 CPH:SA (D) or the soluble groups. Also, there appears to be a biphasic growth trend emerging that may be both time and concentration-dependent, this was not observed for the single drug experiments.

Antimicrobial delivery of RIPE in broth cultures

The combinations of rifampicin/pyrazinamide and isoniazid/ethambutol exhibited time-and concentration-dependent anti-tubercular activity against M. marinum (Figure 5-

2). Currently recommended therapy for active tuberculosis infections is the co-administration of all for drugs. Since the dual delivered drugs yielded combinations that were either antagonistic (RIF/PYR) and synergistic (INH/ETH), all subsequent experiments examined the benefits of NP delivery with the 4 drug cocktail of rifampicin, isoniazid, pyrazinamide, and ethambutol (RIPE). The use of 20:80 CPTEG:CPH with the drug cocktail RIPE (Figure 5-3) provided the most significant benefits of all groups tested.

Initial results revealed that a more extended incubation period was needed for

RIPE drug testing than that used for single or paired combination. The use of the 20:80

CPTEG:CPH nanoparticle provided us a tenfold reduction in MIC when compared to the soluble group (20:80 CPH:SA not shown). A time and concentration-dependent reduction

131 of bacterial viability were observed that maximized killing for the nanoparticle delivery of drugs after an initial lag period of 24–48 h.

Figure 5-2 The killing of M. marinum is enhanced by using nanoparticles loaded with either rifampicin/pyrazinamide or isoniazid/ethambutol in broth. (A) 20:80 CPTEG:CPH RIF/PZA, (B) 20:80 CPTEG:CPH IHN/EMB, (C) 20:80 CPH:SA RIF/PZA, and (D) 20:80 CPH:SA INH/EMB. Colony forming units/ml were enumerated at 48 hrs post-infection and compared with the CFU/ml values for soluble treated bacteria. (Student’s T-test p values * < 0.1, ** < 0.01).

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Figure 5-3 The effectiveness of four drug cocktail against M. marinum. * indicates a significant reduction in bacterial CFU compared to monocyte cultures treated with soluble drug cocktail at p < 0.01.

Intracellular fate of Mycobacterium and NP-RIPE

To test the effectiveness of the nanoparticles on intercellular viability M. marinum was tested with the human monocyte model (THP-1) which has been used (Figure 5-4.) to examine intracellular growth rates of Mycobacterium. Established intracellular infection procedures with Mycobacterium avium subspecies paratuberculosis, employed a multiplicity of infection (MOI) consisting of a bacteria to macrophage ratio low MOI of

1:1 with a single time point of 48 h. A single concentration of either soluble or encapsulated RIPE with either 20:80 CPTEG:CPH or 20:0 CPH:SA. Mycobacteria remain viable

(CFU/ml determination) after 48 h with single soluble drug treatment. Our results showed

133 that at least during the 48 h, THP-1 macrophages in combination with soluble drug cocktail RIPE were only able to provide a 1.69 log CFU (± 1.30) reduction in bacterial counts compared to untreated. In contrast, the nanoparticle 20:80 CPTEG:CPH provided a 6.10 log CFU (± 0) reduction and showed no viable mycobacterium after 48 h.

Figure 5-4 The killing of intracellular M. marinum is enhanced by using nanoparticles loaded with an antibiotic. THP-1 monocytes were infected with M. marinum at an MOI 1:1 to establish a productive intracellular infection. At 24 h, infected cultures were supplemented with 10 μg/mL of either soluble RIPE or encapsulated into polyanhydride particles. Following additional 48 h incubation, nontreated and drug-treated cells were washed and lysed to release intracellular bacteria. The lysate was subsequently serially diluted and plated on solid agar medium. Mean counts of bacteria for untreated cells were 1.1 x 104 CFU/mL. * indicates a significant reduction in bacterial CFU compared to monocyte cultures treated with soluble drug cocktail at (student t- test) p < 0.1.

The 20:80 CPH:SA NP group had variability in the total number of viable bacteria but was still significantly reduced when compared to the soluble group. With the higher MOI concentration of 10:1 an increase in survivability of Mycobacterium within

134 macrophages is was present. Comparing the untreated to the 20:80 CPTEG:CPH NP formulation at 72 h provided a 6.23 log (± 1.74) CFU reduction in bacteria at 48h and 3.40 log CFU (± 0.24) at 72 h. The 20:80 CPH:SA NP formulation provided a 2.86 log CFU

(± 0.11) at 48 h and 3.09 log CFU (± 0.33) at 72 h. At all-time points recovered Myco- bacteria from the soluble treated groups where not statistically significant compared to the untreated.

Intracellular localization of Mycobacterium within THP-1 macrophage

THP-1 macrophages infected with a GFP-expressing M. marinum (GFP) at an MOI of

10:1 where treated with either soluble or NP encapsulated drugs and followed over 72 h.

Field of view (FOV) data provided evidence showing 35% of the macrophage did not internalize mycobacterium-GFP.

There was also evidence of increased cellular death of THP-1 macrophages at the

72 h time points in the untreated and soluble treatment groups, as well as an increased extracellular growth of Mycobacterium. Due to the constitutively expressed GFP in M. marinum, the accurately by which the total number (Figure 4-6) of extracellular and intracellular bacteria counts to determine viability could not be determined. However, the nanoparticle-treated macrophages did show a decrease in the total number of bacteria per cell, and a total reduced number of infected macrophages < 60% was found at 72 h.

Lysosomal localization of M. marinum with polyanhydride nanoparticles particles is essential for delivery of antitubercular. To evaluate the intracellular burden of M. marinum and nanoparticles in THP-1 lysosomal compartments, M. marinum expressing the green fluorescent protein (GFP) residing in THP-1 cells were given NP containing

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2% Rho 20:80 CPTEG:CPH nanoparticles (Figure 5-7) for 48 h and assessed for localization with the lysosomal with LAMP-1 marker.

Figure 5-5 Intracellular targeting of NP in M. marinum infected cells. Monolayers of human monocyte THP-1 cells were infected with M. marinum 10:1 ratio. A) Colony forming units (CFU) were enumerated beginning with a treatment at 48 h postinfection with either soluble drug cocktail (RIF, PYR, EMB, and INH) or equivalent amount encapsulated within 20:80 CPTEG:CPH NP. Intracellular bacterial viability was determined up to 72 hours post-treatment (*p ≤ 0.05, **p ≤ 0.01 Student's t-Test vs. Soluble). (B) Immunofluorescence microscopy of concurrent infected and treated macrophages at 48hrs cultures was imaged to visualize M. marinum (green) with NP (red) and in macrophages (cell nuclei-DNA blue).

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Figure 5-6 Internalization and intracellular viability of mycobacteria with the 20:80 CPH:SA nanoparticle. Monolayers of human monocyte THP-1 cells were infected with M. marinum 10:1 ratio. Colony forming units (CFU) were enumerated at 48 h and 72 h post-infection, survival was calculated compared to CFU/ml soluble drug groups. Susceptibility to 10 μg of Isoniazid, Pyra- zinamide, Ethambutol and Rifampicin compared to formulations of nanoparticles (*p ≤ 0.05 Stu- dent's t-Test vs. Soluble drug groups). Discussion

In the in vitro Mycobacterium broth assays, demonstrated the challenges researchers encounter in development of antitubercular treatments. The use of a single drug of either rifampicin, isoniazid, pyrazinamide or ethambutol in an of themselves are ineffective in killing mycobacteria. The 20:80 CPTEG:CPH provided enhance drug activity when directly compared to the 20:80 CPH:SA formulation. These differences in the killing of M. marinum by the two nanoparticle formulations support the awareness of a tailorable treatment. This tailorability could be adapted for use against multiple pathogens of interest. The 20:80 CPTEG:CPH and 20:80 CPH:SA formulation showed time and

137 concentration-dependent killing as well biphasic killing curve with the used of two antibiotics. The soluble drug treatment group’s had a similar outcome of drug effectiveness as was seen in the single drug treatment. The utilization of a four-drug cocktail of rifampicin, isoniazid, pyrazinamide, and ethambutol in the 20:80 CPTEG:CPH formulation provided superior benefits in drug delivery against M. marinum. The internalization and intracellular viability of M. marinum were significantly reduced with the incorporation of the four drug cocktail encapsulated into the two nanoparticle formulations 20:80

CPTEG:CPH and 20:80 CPH:SA. The unique nature of mycobacteria as an intracellular organism is further enhanced when looking at the survivability in the presence of soluble drugs.We observed a noticeable reduction in the bacterial population of THP-1 infected cells when compared to soluble for both the 20:80 CPTEH:CPH and 20:80 CPH:SA formulation and a time-concentration dependent effect. This effect could be useful in overcoming the natural resistance mechanisms of Mycobacterium through colocalization and increasing the drug cocktail efficacy.

To observe interactions of NP with the host macrophage as well as

Mycobacterium, we performed immunofluorescence staining on infected and treated cell cultures and visualized them by laser scanning confocal microscopy. M. marinum in macrophages treated with nanoparticles were frequently observed to have a substantially reduced GFP expression with an aberrant bacterial morphology typically found in stressed and compromised bacterial membranes. These observations coincide with a 4 fold reduction in intracellular viability measured by CFU enumeration suggesting that internalization of NP reduced intracellular bacterial viability. However, reduced GFP expression and haggard appearance of intracellular bacteria is also observed within

138 macrophages that do not have visible NP within the cell while they can be seen in close proximity outside the cell (Figure 5-7). This suggests that the NP can act from within but also nearby in a paracrine-like fashion.

A caveat to these studies is that it cannot be determined if in fact, the reduced

GFP expressing M. marinum are dead or dying, only that the reduction in CFU coincides with the aberrant bacterial morphology of M. marinum. To this point, not all bacteria within NP treated cells are found associated with lysosomes (Figure 5-7). Normal cellular processing for phagocytosed particles, including nonpathogenic or dead pathogenic bacteria, dictates that the internalized phagosome fuse with Lamp1+ lysosomes to initiate degradation of internalized material. Damaged and dead M. marinum would be more likely to be localized within lysosomes to degrade and process bacterial antigen. The

20:80 CPTEG:CPH nanoparticle provided a reduction in the total infected macrophages over the 48 h but not with the 20:80 CPH:SA or soluble drug-treated cultures. The absence of NP co-localization with all intracellular bacteria could be investigated by incubating M. marinum infected macrophages with 20:80 CPTEG:CPH NP loaded only with rhodamine as we predict drug loaded NP would eliminate bacteria if they did colocalize to the same intracellular compartment. To this end, we observed M. marinum in infected monocytes treated with 20:80 CPTEH:CPH nanoparticles were more likely to be within intracellular compartments that were associated with lysosomes (LAMP1+).

Together, results from this study suggest a promising nanotherapeutic intervention against Mycobacterium is possible through a synergism afforded by encapsulating frontline anti-TB drugs into amphiphilic nanoparticles.

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Figure 5-7 Intracellular growth of M. marinum-GFP within THP-1 macrophages during a 2-day infection period, macrophages were treated with 3.2 μg/ml of the drug as either soluble or encapsulated into nanoparticles.THP-1 macrophages were plated on to glass- cover slips in a 24 well tissue culture plate and infected at an MOI of 10:1 with M. marinum pGFPHYG2. 2% Rho B-loaded CPTEG:CPH nanoparticles (RED), M. marinum GFPHYG2 (green) at an infectious dose of 10:1 for 48 h, THP-1 were stained with the anti-LAMP-1 antibody (magenta), Nucleus staining DAPI (blue). (A) Soluble 24 h,(B)

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Figure 5-7 (continued) NP 24 h, (C) Soluble 48 h, and (D) NP 48. Scale bar indicates 10μm. White arrowheads note intracellular bacteria based on proximity to intracellular features and location of plasma membrane from brightfield analysis (not shown). Yellow arrowheads point out extracellular bacteria. The intensity of GFP from M. marinum was consistently observed to be lower in NP-treated at 48 hours (panel D) than soluble (panel C). Conclusion

This work is the first reported use of a polyanhydride nanoparticle against

Mycobacterium. We observed through this study the importance of developing a delivery platform that can overcome the natural resistance mechanisms of Mycobacteria. We did not observe significant killing of M. marinum at the higher concentrations with the use of the soluble drugs rifampicin, isoniazid, pyrazinamide and ethambutol. This deficiency strengthens the hypothesis that use of the polyanhydride nanoparticle delivery vehicle increases the efficacy of the antitubercular drugs. The encapsulation of the four drugs into polyanhydride nanoparticles significantly improved intracellular delivery targetting established Mycobacterium infections in vitro resulting in improved drug efficacy with dose sparing benefits.

References

1. Organization, W.H. Global tuberculosis report 2017. 2017 [cited 2017 December 01]; Available from: http://www.who.int/tb/publications/global_report/en/.

2. Klopper, M., et al., Emergence and Spread of Extensively and Totally Drug- Resistant Tuberculosis, South Africa. Emerging Infectious Diseases, 2013. 19(3): p. 449-455.

3. Saravanan, M., et al., Review on emergence of drug-resistant tuberculosis (MDR & XDR-TB) and its molecular diagnosis in Ethiopia. Microb Pathog, 2018. 117: p. 237-242.

141

4. Berrada, Z.L., et al., Rifabutin and rifampin resistance levels and associated rpoB mutations in clinical isolates of Mycobacterium tuberculosis complex. Diagn Microbiol Infect Dis, 2016. 85(2): p. 177-81.

5. Jamieson, F., et al., Profiling of rpoB Mutations and MICs for Rifampin and Rifabutin in Mycobacterium tuberculosis. Journal of Clinical Microbiology, 2014. 52(6): p. 2157.

6. Namouchi, A., et al., Phenotypic and genomic comparison of Mycobacterium aurum and surrogate model species to Mycobacterium tuberculosis: implications for drug discovery. BMC Genomics, 2017. 18: p. 530.

7. Dharra, R., et al., Rational design of drug-like compounds targeting Mycobacterium marinum MelF protein. PLoS ONE, 2017. 12(9): p. e0183060.

8. Forrellad, M.A., et al., Virulence factors of the Mycobacterium tuberculosis complex. Virulence, 2013. 4(1): p. 3-66.

9. Stinear, T.P., et al., Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis. Genome Res, 2008. 18(5): p. 729-41.

10. Ouertatani-Sakouhi, H., et al., Inhibitors of Mycobacterium marinum virulence identified in a Dictyostelium discoideum host model. PLoS One, 2017. 12(7): p. e0181121.

11. El-Etr, S.H., et al., Identification of Two Mycobacterium marinum Loci That Affect Interactions with Macrophages. Infection and Immunity, 2004. 72(12): p. 6902-6913.

12. Liu, Y., et al., Identification of a novel inhibitor of isocitrate lyase as a potent antitubercular agent against both active and non-replicating Mycobacterium tuberculosis. Tuberculosis (Edinb), 2016. 97: p. 38-46.

13. Lienard, J. and F. Carlsson, Murine Mycobacterium marinum Infection as a Model for Tuberculosis. Methods Mol Biol, 2017. 1535: p. 301-315.

14. Kenyon, A., et al., Active nuclear transcriptome analysis reveals inflammasome- dependent mechanism for early neutrophil response to Mycobacterium marinum. Sci Rep, 2017. 7(1): p. 6505.

15. Patel, S.S., et al., Necrotizing Soft Tissue Infection Occurring after Exposure to Mycobacterium marinum. Case Rep Infect Dis, 2014. 2014: p. 702613.

142

16. Griffith, D.E., et al., An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med, 2007. 175(4): p. 367-416.

17. Rallis, E. and E. Koumantaki-Mathioudaki, Treatment of Mycobacterium marinum cutaneous infections. Expert Opin Pharmacother, 2007. 8(17): p. 2965- 78.

18. Lewis, F.M., B.J. Marsh, and C.F. von Reyn, Fish tank exposure and cutaneous infections due to Mycobacterium marinum: tuberculin skin testing, treatment, and prevention. Clin Infect Dis, 2003. 37(3): p. 390-7.

19. Rodrigues, L., et al., Ethidium bromide transport across Mycobacterium smegmatis cell-wall: correlation with antibiotic resistance. BMC Microbiol, 2011. 11: p. 35.

20. Verschoor, J.A., M.S. Baird, and J. Grooten, Towards understanding the functional diversity of cell wall mycolic acids of Mycobacterium tuberculosis. Prog Lipid Res, 2012. 51(4): p. 325-39.

21. Lee, S.Y., et al., Effect of amikacin on cell wall glycopeptidolipid synthesis in Mycobacterium abscessus. J Microbiol, 2017. 55(8): p. 640-647.

22. Li, Y., et al., Design and synthesis of novel cell wall inhibitors of Mycobacterium tuberculosis GlmM and GlmU. Carbohydr Res, 2011. 346(13): p. 1714-20.

23. Bhat, Z.S., et al., Cell wall: A versatile fountain of drug targets in Mycobacterium tuberculosis. Biomed Pharmacother, 2017. 95: p. 1520-1534.

24. Singh, G., et al., Cell Wall Associated Factors of Mycobacterium tuberculosis as Major Virulence Determinants: Current Perspectives in Drugs Discovery and Design. Curr Drug Targets, 2017. 18(16): p. 1904-1918.

25. Bredehorn, T., et al., Morphological and functional changes due to drug-induced lysosomal storage of sulphated glycosaminoglycans in the rat retina. Graefes Arch Clin Exp Ophthalmol, 2001. 239(10): p. 788-93.

26. Fischer, J., H. Lullmann, and R. Lullmann-Rauch, Drug-induced lysosomal storage of sulphated glycosaminoglycans. Gen Pharmacol, 1996. 27(8): p. 1317- 24.

27. Sakhrani, N.M. and H. Padh, Organelle targeting: third level of drug targeting. Drug Des Devel Ther, 2013. 7: p. 585-99.

143

28. Anguru, M.R., et al., Novel drug targets for Mycobacterium tuberculosis: 2- heterostyrylbenzimidazoles as inhibitors of cell wall protein synthesis. Chem Cent J, 2017. 11(1): p. 68.

29. Bacon, J., et al., Non-replicating Mycobacterium tuberculosis elicits a reduced infectivity profile with corresponding modifications to the cell wall and extracellular matrix. PLoS One, 2014. 9(2): p. e87329.

30. Cheung, J.P., et al., Mycobacterium marinum infection of the hand and wrist. J Orthop Surg (Hong Kong), 2012. 20(2): p. 214-8.

31. Binnebose, A.M., et al., Polyanhydride Nanoparticle Delivery Platform Dramatically Enhances Killing of Filarial Worms. PLoS Negl Trop Dis, 2015. 9(10): p. e0004173.

32. Chazel, M., et al., Evaluation of the SLOMYCO Sensititre((R)) panel for testing the antimicrobial susceptibility of Mycobacterium marinum isolates. Ann Clin Microbiol Antimicrob, 2016. 15: p. 30.

33. Torres, M.P., et al., Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. J Biomed Mater Res A, 2006. 76(1): p. 102-10.

34. Domb, A.J., et al. Polyanhydrides: Synthesis and characterization. 1993. Berlin, Heidelberg: Springer Berlin Heidelberg.

35. Kipper, M.J., et al., Design of an injectable system based on bioerodible polyanhydride microspheres for sustained drug delivery. Biomaterials, 2002. 23(22): p. 4405-12.

36. Torres, M.P., et al., Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. Journal of biomedical materials research. Part A, 2006. 76(1): p. 102.

37. Haughney, S.L., et al., Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles. Acta Biomater, 2013. 9(9): p. 8262-71.

38. Ulery, B.D., et al., Design of a protective single-dose intranasal nanoparticle- based vaccine platform for respiratory infectious diseases. PLoS One, 2011. 6(3): p. e17642.

39. Huntimer, L., et al., Evaluation of biocompatibility and administration site reactogenicity of polyanhydride-particle-based platform for vaccine delivery. Adv Healthc Mater, 2013. 2(2): p. 369-78.

144

40. Parish, T. and N.G. Stoker, Electroporation of Mycobacteria, in Electroporation Protocols for Microorganisms, J.A. Nickoloff, Editor. 1995, Humana Press: Totowa, NJ. p. 237-252.

41. Peñuelas-Urquides, K., et al., Measuring of Mycobacterium tuberculosis growth. A correlation of the optical measurements with colony forming units. Brazilian Journal of Microbiology, 2013. 44(1): p. 287-289.

42. Bellaire, B.H., R.M. Roop, and J.A. Cardelli, Opsonized Virulent Brucella abortus Replicates within Nonacidic, Endoplasmic Reticulum-Negative, LAMP-1- Positive Phagosomes in Human Monocytes. Infection and Immunity, 2005. 73(6): p. 3702-3713.

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CHAPTER 6 . AMPHIPHILIC POLYANHYDRIDE NANOPARTICLE DRUG DELIVERY PLATFORM EFFECTIVELY KILLS MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS

For publication in a journal of veterinary medicine

Andrea M. Binnebose1, Shannon L. Haughney2, Balaji Narasimhan2 and Bryan Bellaire1

1 Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, 50011 2 Department of Chemical and Biological Engineering, Iowa State University, Ames, 50011 * To whom all correspondence must be addressed

E-mail: [email protected]; [email protected]; Phone: (515) 294-1006, [email protected]; Phone:(515) 294-8019

Abstract

The causative agent of Johne's disease and Crohn’s is a facultative intracellular enteric bacteria known as Mycobacterium avium subspecies paratuberculosis. As with

Mycobacterium species, the cause chronic and persistent infections facilitated by intracellular survival in host cells that prevent exposure to soluble antimicrobial agents used to treat the disease. Formulation of antimicrobial agents within bioerodable nanoparticles is a means to target infected cells to eliminate intracellular resident bacterial pathogens including Mycobacterium species. The antitubercular drug cocktail containing rifampicin, isoniazid pyrazinamide and ethambutol were successfully encapsulated into amphiphilic nanoparticles synthesized with varying amounts of the hydrophobic carboxyphenoxyhexane polymer. Results showed that polyanhydride nanoparticles are

146 internalized efficiently by macrophages, traffic to endosomes, and release high concentrations of antituberculosis drugs intracellularly. We demonstrated that drugs delivered by polyanhydrides killed Mycobacteria within macrophages significantly more effectively than standard administration of soluble drugs. This data indicates that polyanhydride nanoparticles provide a versatile platform that can be optimized through alternating polymer formulation, changing the loading percentile of each drug and intracellular release of specific drugs for the treatment of Mycobacteria.

Introduction

Mycobacterium avium subspecies paratuberculosis is the causative agent of chronic granulomatous enteritis in ruminants called Johne's disease (JD) [1] as well as suspected in the human inflammatory bowel diseases Crohn’s (CD) and Ulcerative Coli- tis (UC) [2-6]. Mycobacterium avium subspecies paratuberculosis (MAP) is a nontuberculosis mycobacterium, of the Mycobacterium avium complex most often associated with the small intestinal lamina propria that is eventually fatal in many cases [7, 8]. The infection in ruminants is most commonly transmitted through the fecal-oral route due to contaminated closed confinement, pasture, soil, and water, as well as vertical intrauterine transfer [9]. The effects of JD on the U .S. dairy industry accounts for upwards of $220 million per year, with 2 out of every three farms being seropositive [10]. Like most mycobacterium infection symptomology does not occur until many months after initial expose and treatment in ruminates is not suggested.

Source of human contamination is believed to be a direct result of bacterial shedding into drinking water, milk and in even meat [11-13]. The unique thick lipid cell wall allows the bacteria to survive pasteurization [14].The occurrences of the MAP being

147 found in individual testing positive for CD has been shown to exceed 59% [5, 6] but testing is limited and can be unreliable [11]. Individuals testing seropositive for the MAP may undergo extensive extended drug therapy (<5 weeks) [15] using the standard antituberculous rifampicin, isoniazid, ethambutol, and pyrazinamide [16] as well as clythromycin [17]. Treatment of infected individuals caused by Mycobacterium avium complex remains a challenge since these organisms are resistant to the majority of antituberculous drugs [16, 18].

Poly anhydride nanoparticles improve the antimicrobial activity of anti- tuberculocidal drugs against Mycobacterium marinum in broth as well as co-localize with the bacteria inside of host phagocytes. Polyanhydrides nanoparticles are solid, surface eroding particles that encapsulate small molecules and proteins which have been shown to become an integral component of the particles [19, 20]. This next-generation platform has demonstrated the capabilities in vitro to become a multiple drug delivery system with the ability to increase the efficacy of drugs against M. marinum. The ability of the nanoparticles to slowly erode and release the cargo molecule in a controlled manner allows for targeted antimicrobial activity. Factors that contribute to the difficulty in treating MAP infections are the hydrophobic mycolic acids of the outer membrane, slow growth, and establishing unique intracellular niche are shared by all pathogenic Mycobacterial species. In this study, we will demonstrate the antimicrobial activity of polyanhydride nanoparticles in delivering rifampicin, isoniazid, pyrazinamide, and ethambutol.

We tested the growth rate of M. paratuberculosis ATCC 19698 with turbidly testing and looked at the effectiveness of polyanhydride nanoparticles in co-localization assays in the human macrophage THP-1 and the murine macrophage RAW 264.7. We

148 will look at survivability of MAP and nanoparticle interactions with both cell lines. We will also look at the polymers and copolymers based on CPTEG, CPH, and SA formulation in their effectiveness in drug delivery and interaction with the MAP. Through this and previous work, we have come to recognize the enormous potential in the use of polyanhydride in medicine, through their unique ability in encapsulating small molecules and as well as increasing the efficacy of drugs.

Materials and methods

Nanoparticles

Science (St. Louis, MO). Rifampicin, isoniazid, pyrazinamide and ethambutol were also purchased from Sigma Life Science (St. Louis, MO).

149

Polymer Synthesis

Synthesis of the monomers used, 1,6-bis(p-carboxyphenoxyhexane) (CPH) and

1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) was performed as described elsewhere [21]. 20:80 molar copolymers of CPTEG and CPH and CPH and SA were synthesized through melt condensation polymerization, as previously described in detail [22-24]

. Molecular weight was confirmed using 1H NMR.

Nanoparticle Fabrication

Rifampicin, isoniazid, pyrazinamide and ethambutol-loaded nanoparticles of

20:80 CPH:SA and 28:80 CPTEG:CPH were fabricated through solid/oil/oil nanoprecipitation, as previously described [25-27]. Drug combinations Rifampicin and pyrazinamide, each at 5% (w/w), with Rhodamine B at 2% (w/w) were added to the polymer.

Drug combinations, isoniazid and ethambutol each at 5% (w/w), with Rhodamine B at

2% (w/w) were added to the polymer. The solid drug and polymer mixture was then dissolved in methylene chloride at a concentration of 20 mg/mL followed by rapid precipitation into the antisolvent, pentane. The polyanhydride nanoparticles were then collected using vacuum filtration. The drug-loaded particles were characterized for size and morphology by scanning electron microscopy (SEM, FEI Quanta SEM, Hillsboro, OR).

Bacterial strains and growth conditions

Mycobacterium avium paratuberculosis (ATCC 19698) was propagated at 37°C either in Middlebrook 7H9 broth, containing 10% oleic acid-albumin-dextrose-catalase,

0.05% Tween 80 (Sigma), ferric mycobactin J (Allied Monitor) and amphotericin B

(Sigma), or on Middlebrook 7H10 agar containing 10% oleic acid-albumin-dextrose- catalase, ferric mycobactin J (Allied Monitor) and amphotericin B (Sigma) . Frozen

150 stocks were prepared by growing bacteria in 7H9 broth to mid-log phase approximately

3.5 weeks or until optical density at 600 nm [OD600], ∼1, collecting cells by centrifugation, washing once with phosphate-buffered saline containing 0.05% Tween 80

(PBST), adding glycerol to 20%, and storing in aliquots at −80°C. Bacterial stocks were titered by plating PBST-diluted bacterial suspensions on 7H10 agar and scoring colonies after eight weeks at 37°C 5%CO2 humidified chamber.

Cells and culture conditions

The human monocytic cell line THP-1 was cultured in RPMI 1640 medium 10%

× 106 cells/ml, resuspended in fresh medium supplemented with 5 nM phorbol myristic acid (PMA) [28] to differentiate the cells into adherent monocytes, and placed into 96 well tissue culture plates. After overnight culture in the presence of PMA, adherent cells were washed three times gently with phosphate-buffered saline (PBS; pH 7.4) and incubated for an additional 24 h in complete RPMI 1640 medium with no PMA. The monocytic adherent murine macrophage cell line RAWs 264.7 was cultured in DMEM medium with 10% (v/v) heat-inactivated fetal bovine serum. Cultures where maintain at similar cell density as described above.

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Bacterial turbidity growth curve

At each time point (3d), an aliquot of 1 ml of a 15 ml conical tube was disrupted mechanically with sterile 2mm stainless steel beads by vortexing. 900 microliters of the bacterial suspension were added to 8.1 ml of phosphate-buffered saline (PBS) (pH 7.2) was added slowly and vortexed to obtain a homogeneous cell suspension. One milliliter was transferred to polystyrene cuvette, and three separate values were recorded via a spectrometer (Bio-Rad), and OD 600 values were recorded over 28 days.

Broth microdilution antimicrobial assay

Antibiotic testing against M. marinum was determined in 7H9 broth by the standard microdilution method. Briefly, 0.1-ml of a 0.1 mg ml-1 dilution of previously mentioned drugs were added to a 96-well plate. Control wells included medium and culture controls. One hundred microliters of culture (at 0.5 x 106 CFU/ml) were added to wells except for the medium control wells. The plates were incubated at 37°C for up to 72 h.

Following each incubation time intervals, twenty microlitres of each bacterial suspension were serially diluted in PBS and aliquots of triplicate were plated onto supplemented

Middlebrook 7H10. The plates were sealed and incubated for 6-10 weeks at 37°C

5%CO2. Experiments were conducted in triplicate and the standard deviation is reported on graphs unless otherwise stated.

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Intracellular survival of Mycobacterium

THP-1 cells and Raw264.7 used for evaluation of the intracellular survival of M. avium paratuberculosis were treated similarly as previously described in 96-well flat-bottom tissue culture plates. Bacterial suspensions were prepared and generated by scraping

36 d cultures of the M. avium paratuberculosis grown on supplemented Middlebrook

7H10 agar into screw-cap microfuge tubes containing supplemented 7H9 media and incubated for seven days. Pellets of bacteria were resuspended through mechanical disruption and numbers of bacteria present in the suspensions were determined by optical density

OD 600 nm measurements [29]. The suspensions were diluted to desired amounts using complete RPMI 1640 or DMEM medium to maintain a constant multiplicity of infection with slight variations in numbers of target monocytes. Suspensions of bacteria were added to monocyte monolayers at a multiplicity of infection (bacteria/monocyte ratio) of

10:1. Tissue culture plates were gently agitated by hand and then centrifuged at 4°C for

10 min at 270 x g. Moreover, transferred to an incubator for 90 min 37°C with 5% CO2 to allow for phagocytosis of bacteria. Monolayers were washed three times with PBS to remove any remaining nonadherent bacteria. Fresh RPMI-1640 or DMEM complete media was added following the last washing. The viability of intracellular Mycobacterium was determined by lysing monocytes with 0.1% deoxycholate, diluting suspensions in

PBS, and plating aliquots in triplicate on supplemented Middlebrook 7H10 medium. The plates were sealed and incubated for 6-10 weeks at 37°C 5%CO2. Total cfu/ml of bacterial survival at 24, 48 and 72 h were calculated based on the number of internalized

153 bacteria detected at 2 h postinfection which represents 100% of internalized bacteria. Ex- periments were conducted in triplicate and the standard deviation is reported on graphs unless otherwise stated.

Statistical Analysis

The statistical software GraphPad Prism (v. 7.04) was used to analyze the data.

Student’s t-test was used to determine statistical significance among treatments and p<

0.05 was considered significant.

Results

The in vitro susceptibility of human and bovine origin M. paratuberculosis is regulated by initial bacterial concentration and is a critical factor in drug susceptibility testing of slow-growing mycobacteria. The growth of M. paratuberculosis ATCC 19698 was recorded by measuring changes in the optical density of broth cultures. The initial bacterial concentrations test began with a 104, 105, and 106 CFU/ML. We followed the growth curve over 28 days by optical density and observed an average replication rate of

<8 days (data not shown). The ability to accurately determine a correct replication rate was inherently flawed due to the natural clumping of MAP over the course of the assay.

Other assays corroborate this same issue and have demonstrated that the method is not reliable [30]. In total, we performed 12 culture inhibition experiments that included the use of soluble and or encapsulated rifampicin, isoniazid, ethambutol, and pyrazinamide. In every case where antibiotics were studied for its effect on MAP, time and delivery method where the dependent variables.

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Cultures treated with a drug combination of rifampicin/pyrazinamide (Figure 5-1), where cultures contained 10 μg of each drug per ml of MAP culture. Equivalent amounts of the drug cocktail were given in either soluble form or encapsulated into nanoparticles to assess antimicrobial activity measured by direct plating of cultures to quantify bacterial viability.

At 48 hours, untreated MAP grew to 3.44 log CFU while soluble reduced bacterial viability to 1.55 log CFU (± 0.35) demonstrating a significant difference

(p<0.001). Cultures treated with either NP formulation containing RIF/PZA produced no viable bacteria demonstrating a significant reduction from soluble antimicrobial activity

(p<0.01).

Figure 6-1 Antimicrobial activity of RIF/PZA NP against MAP. Mycobacterium avium subspecies paratuberculosis was tested for susceptibility to 10 μg of Pyrazinamide and Rifampicin. The graph depicts the comparison between two soluble drugs to 20:80 CPTEG:CPH and 20:80 CPH:SA dual loaded nanoparticles. Significance being *p ≤ 0.05 Student's t-Test vs. Soluble drug groups.

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In contrast to the RIF/PZA combination, soluble INH/EMB treatment at the same

10 µg concentration, did not have antimicrobial activity (Figure 6-2). The 20:80

CPTEG:CPH group had a total 3.22 log CFU (± 0) reduction in bacterial population with no viable growth detected. Whereas the 20:80 CPH:SA group also had a (p < 0.05) significant total 1.45 log CFU (± 0.90) FOR ALL OF THESE NUMBERS reduction in bacterial population but growth was evident.

Figure 6-2 Antimicrobial activity of INH/EMB NP against MAP. Mycobacterium avium subspecies paratuberculosis was tested for susceptibility to 10 μg of Isoniazid and Ethambutol. The graph depicts the comparison between two soluble drugs to 20:80 CPTEG:CPH and 20:80 CPH:SA dual loaded nanoparticles. Significance being *p ≤ 0.05 Student's t-Test vs. Soluble drug groups. The combined therapeutic approach of rifampicin/pyrazinamide and isoniazid/ethambutol (Figure 6-3) was tested with the four-drug combination at a single dose concentration and monitored over 48 h. The 20:80 CPTEG:CPH nanoparticle group displayed the most significant bactericidal activity against Mycobacteria with a total 3.50 log

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CFU/mL reduction in bacterial population. The bactericidal activity was not observed with the 20:80 CPH:SA nanoparticles which provided only a bacteriostatic effect of 1.16 log CFU (± 1.10) reduction in bacterial population compared to untreated. Both nanoparticle groups where significantly different than the soluble group which had only minimal inhibition of 0.652 log CFU (± 0.12) of the bacterial population.

Figure 6-3 Antimicrobial activity of RIP/PZA/INH/EMB NP against MAP. Mycobacterium avium subspecies paratuberculosis was tested for susceptibility to 10 μg to Isoniazid, Pyra- zinamide, Ethambutol, and Rifampicin. (*p ≤ 0.05 Student's t-Test vs. Soluble drug groups).

Several macrophage models have been used to study mycobacteria infections.

These include primary macrophages, mainly human monocyte-derived macrophage

(THP-1) or murine J774 and RAW 264.7. We conducted a short course therapy against

THP-1 and RAW 264.7 macrophages to determine an adequate infectious model for use in slow-growing mycobacteria. Concurrently, we also tested a single dose of the four-

157 drug combination RIPE in soluble or encapsulated in nanoparticles. We observed that the

THP-1 was simultaneously effective in conjunction with the 20:80 CPTEG:CPH nanoparticle within 48 h. The 20:80 CPTEG:CPH nanoparticle group (Figure 6-4) provided a

3.792 log CFU (± 0) inhibition within the bacterial population over the 48 h and the

20:80 CPH:SA 2.062 logs CFU (± 1.09) compared to the soluble.

Figure 6-4 Internalization and intracellular viability. Monolayers of human monocyte THP-1 cells were infected with Mycobacterium avium subspecies paratuberculosis 10:1 ratio. Colony forming units (CFU) were enumerated at 48 h postinfection, survival was calculated compared to CFU/ml soluble drug groups. Susceptibility to 10 μg of Isoniazid, Pyrazinamide, Ethambutol, and Rifam- picin compared to formulations of PANs. (*p ≤ 0.05 Student's t-Test vs. Soluble drug groups)

The use of the murine macrophage RAW 264.7 showed a more robust bacterial population in direct comparison to the THP-1 macrophages. We observed the natural wean in growth at 24 h and subsequently observed exponential growth. This is consistent with what is reported with the use of murine macrophages. The 20:80 CPTEG:CPH NPs

(Figure 6-5) reduced the bacterial burden by 1.674 log CFU (± 0.08) at 48 h and 3.515 log CFU (± 1.69) at 72 h compared to the non-treated. The solubly treated MAP at 48 h

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0.054 log CFU (± 0.18) and 0.609 log CFU (± 0.14) at 72 h. The CPH:SA NPs 1.368 log

CFU (± 0.15) at 48 h and 2.007 log CFU (± 0.33) at 72 h (Figure 5-6). The use of the four-drug combination RIPE for the soluble treated macrophages did not provide addition bactericidal activity against M. paratuberculosis.

Figure 6-5 Intracellular viability MAP within murine macrohpages following treatment with RIF/PZA/INH/EMB CPTEG:CPH NP. Monolayers of murine macrophage 267.4 cells were infected with Mycobacterium avium subspecies paratuberculosis 10:1 ratio. Colony forming units (CFU) were enumerated at 24,48 and 72 h postinfection, survival was calculated compared to CFU/ml soluble drug groups. Susceptibility to 10 μg of Isoniazid, Pyrazinamide, Ethambutol, and Rifampicin compared to 20:80 CPTEG:CPH polyanhydride nanoparticle. Standard error is denoted on graphs (*p ≤ 0.05 Student's t-Test vs. Soluble drug groups.

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Discussion

We demonstrated the ability of polyanhydride nanoparticles to improve killing of intracellular M. paratuberculosis. We also demonstrated that use of polyanhydride nanoparticles provides greater internalized efficacy by macrophages, increased trafficking to

endosomes, and releases higher concentrations of antituberculosis drugs intracellularly.

We have shown that the four-drug combination rifampicin, isoniazid, pyrazinamide and

ethambutol drugs delivered by polyanhydride nanoparticles killed Mycobacteria within

Figure 6-6 Intracellular viability MAP within murine macrohpages following treatment with RIF/PZA/INH/EMB CPH:SA NP Monolayers of murine macrophage 267.4 cells were infected with Mycobacterium avium subspecies paratuberculosis 10:1 ratio. Colony forming units (CFU) were enumerated at 24, 48 and 72 h postinfection, survival was calculated compared to CFU/ml soluble drug groups. Susceptibility to 10 μg of Isoniazid, Pyrazinamide, Ethambutol, and Rifam- picin compared to 20:80 CPH:SA polyanhydride nanoparticle. Standard error is denoted on graphs (*p ≤ 0.05 Student's t-Test vs. Soluble drug groups)

160 macrophages significantly more effectively than an equivalent amount of free drug. We also demonstrated the importance of utilizing an appropriate macrophage cell line in testing against slow vs. fast-growing mycobacteria. Our observation was consistent with what is reported [31] with the use of murine macrophages vs. human macrophages. We found that the THP-1 macrophage responded very strongly to infection whereas the response of RAW 267.4 to M. paratuberculosis infection is weaker in the magnitude of suppression, at least regarding the overall length of our study. This data demonstrates that there may be a more suitable carrier for drug delivery to treat slow-growing intracellular

Mycobacterium pathogens. This study outlines an approach that allows for a multiple drug delivery platform showing increased efficacy and intracellular pathogen targeting.

References

1. Stevenson, K., Genetic diversity of Mycobacterium avium subspecies paratuberculosis and the influence of strain type on infection and pathogenesis: a review. Vet Res, 2015. 46: p. 64.

2. Bernstein, C.N., et al., Population-Based Case Control Study of Seroprevalence of Mycobacterium paratuberculosis in Patients with Crohn's Disease and Ulcerative Colitis. Journal of Clinical Microbiology, 2004. 42(3): p. 1129-1135.

3. Collins, M.T., et al., Results of Multiple Diagnostic Tests for Mycobacterium avium subsp. paratuberculosis in Patients with Inflammatory Bowel Disease and in Controls. Journal of Clinical Microbiology, 2000. 38(12): p. 4373-4381.

4. Lee, A., et al., Association of Mycobacterium avium subspecies paratuberculosis with Crohn Disease in pediatric patients. J Pediatr Gastroenterol Nutr, 2011. 52(2): p. 170-4.

5. Pierce, E.S., Could Mycobacterium avium subspecies paratuberculosis cause Crohn’s disease, ulcerative colitis…and colorectal cancer? Infectious Agents and Cancer, 2018. 13: p. 1.

6. Timms, V.J., et al., The Association of Mycobacterium avium subsp. paratuberculosis with Inflammatory Bowel Disease. PLoS ONE, 2016. 11(2): p. e0148731.

161

7. Whittington, R.J., et al., Case definition terminology for paratuberculosis (Johne's disease). BMC Vet Res, 2017. 13(1): p. 328.

8. Bates, A., et al., The effect of sub-clinical infection with Mycobacterium avium subsp. paratuberculosis on milk production in a New Zealand dairy herd. BMC Vet Res, 2018. 14(1): p. 93.

9. Whittington, R.J., et al., Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: IS900 restriction fragment length polymorphism and IS1311 polymorphism analyses of isolates from animals and a human in Australia. J Clin Microbiol, 2000. 38(9): p. 3240-8.

10. United States Department of Agriculture. 2017 Aug 18, 2017 03/18/2018]; Johne's Disease]. Available from: https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/nvap/NVAP-Reference- Guide/Control-and-Eradication/Johnes-Disease.

11. Patel, A. and N. Shah, Mycobacterium avium subsp paratuberculosis--incidences in milk and milk products, their isolation, enumeration, characterization, and role in human health. J Microbiol Immunol Infect, 2011. 44(6): p. 473-9.

12. Pribylova, R., et al., Mycobacterium avium subsp. Paratuberculosis and the expression of selected virulence and pathogenesis genes in response to 6 degrees C, 65 degrees c and ph 2.0. Braz J Microbiol, 2011. 42(2): p. 807-17.

13. Pickup, R.W., et al., Mycobacterium avium subsp. paratuberculosis in lake catchments, in river water abstracted for domestic use, and in effluent from domestic sewage treatment works: diverse opportunities for environmental cycling and human exposure. Appl Environ Microbiol, 2006. 72(6): p. 4067-77.

14. Slater, N., et al., Impact of the shedding level on transmission of persistent infections in Mycobacterium avium subspecies paratuberculosis (MAP). Vet Res, 2016. 47: p. 38.

15. Pribylova, R., et al., Effect of short- and long-term antibiotic exposure on the viability of Mycobacterium avium subsp. paratuberculosis as measured by propidium monoazide F57 real time quantitative PCR and culture. Vet J, 2012. 194(3): p. 354-60.

16. Krishnan, M.Y., E.J.B. Manning, and M.T. Collins, Comparison of three methods for susceptibility testing of Mycobacterium avium subsp. paratuberculosis to 11 antimicrobial drugs. Journal of Antimicrobial Chemotherapy, 2009. 64(2): p. 310-316.

162

17. Hermon-Taylor, J., Treatment with drugs active against Mycobacterium avium subspecies paratuberculosis can heal Crohn's disease: more evidence for a neglected Public Health tragedy. Digestive and Liver Disease. 34(1): p. 9-12.

18. Piersimoni, C., et al., Evaluation of a New Method for Rapid Drug Susceptibility Testing of Mycobacterium avium Complex Isolates by Using the Mycobacteria Growth Indicator Tube. Journal of Clinical Microbiology, 1998. 36(1): p. 64-67.

19. Petersen, L.K., et al., Amphiphilic polyanhydride nanoparticles stabilize Bacillus anthracis protective antigen. Mol Pharm, 2012. 9(4): p. 874-82.

20. Walkey, C.D., et al., Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake. J Am Chem Soc, 2012. 134(4): p. 2139-47.

21. Torres, M.P., et al., Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. J Biomed Mater Res A, 2006. 76(1): p. 102-10.

22. Domb, A.J., et al. Polyanhydrides: Synthesis and characterization. 1993. Berlin, Heidelberg: Springer Berlin Heidelberg.

23. Kipper, M.J., et al., Design of an injectable system based on bioerodible polyanhydride microspheres for sustained drug delivery. Biomaterials, 2002. 23(22): p. 4405-12.

24. Torres, M.P., et al., Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. Journal of biomedical materials research. Part A, 2006. 76(1): p. 102.

25. Haughney, S.L., et al., Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles. Acta Biomater, 2013. 9(9): p. 8262-71.

26. Ulery, B.D., et al., Design of a protective single-dose intranasal nanoparticle- based vaccine platform for respiratory infectious diseases. PLoS One, 2011. 6(3): p. e17642.

27. Huntimer, L., et al., Evaluation of biocompatibility and administration site reactogenicity of polyanhydride-particle-based platform for vaccine delivery. Adv Healthc Mater, 2013. 2(2): p. 369-78.

28. Bellaire, B.H., R.M. Roop, and J.A. Cardelli, Opsonized Virulent Brucella abortus Replicates within Nonacidic, Endoplasmic Reticulum-Negative, LAMP-1- Positive Phagosomes in Human Monocytes. Infection and Immunity, 2005. 73(6): p. 3702-3713.

163

29. Peñuelas-Urquides, K., et al., Measuring of Mycobacterium tuberculosis growth. A correlation of the optical measurements with colony forming units. Brazilian Journal of Microbiology, 2013. 44(1): p. 287-289.

30. Elguezabal, N., et al., Estimation of Mycobacterium avium subsp. paratuberculosis Growth Parameters: Strain Characterization and Comparison of Methods. Applied and Environmental Microbiology, 2011. 77(24): p. 8615- 8624 31. Andreu, N., et al., Primary macrophages and J774 cells respond differently to infection with Mycobacterium tuberculosis. Scientific Reports, 2017. 7: p. 42225.

CHAPTER 7 . NANOBASED DRUG DELIVERY ENHANCES THE ACTIVITY OF ANTIBIOTIC COCKTAIL AGAINST MULTI-DRUG RESISTANT MYCOBACTERIUM

Manuscript to be submitted to a PLOS Pathogens

Andrea M. Binnebose1, Adam S. Mullis 2, Shannon L. Haughney2, Balaji Narasimhan2*, and Bryan H. Bellaire1* 1 Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, 50011 2 Department of Chemical and Biological Engineering, Iowa State University, Ames, 50011 * To whom all correspondence must be addressed

E-mail: [email protected]; [email protected]; Phone: (515) 294-1006, nbala- [email protected]; Phone:(515) 294-8019

Abstract

Inadequate dosing and incomplete treatment regimens, coupled with the ability of the tuberculosis bacilli to cause latent infections that are tolerant of currently used drugs, have fueled the rise of multidrug-resistant (MDR-TB) and extensively drug-resistant

(XDR-TB) tuberculosis. Treatment of both MDR-TB and XDR-TB infections is a significant clinical challenge that has few viable or effective solutions; therefore patients face a poor prognosis and years of therapy. This research focuses on the development of a new drug delivery platform utilizing polyanhydride nanoparticles in combination with the anti-tubercular drugs rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) as a drug cocktail (RIPE) that have the potential for treating MDR-TB and XDR-TB. This research highlights the use of the use of the nanoparticle platforms 165 strengths to as it pertains to, in vitro and in vivo efficacy. Herein, we demonstrate the current challenges in the treatment of tuberculosis and how the use of a nanoparticle- based drug delivery platform can adapt to these difficulties, with an emphasis on

Mycobacterium susceptibility to first-line drugs.

Introduction

Many intracellular pathogens gain initial entry through the host mucosal surfaces, especially those of the respiratory tracts. Transmission can be primarily a result of person-to-person contact and aerosolization of droplets containing bacteria that are small enough to enter the alveoli [1]. Most facultative intracellular bacteria cause chronic or re- current infections, as is the case with Mycobacterium tuberculosis. M. tuberculosis

(MTB) have developed several strategies to prevent their killing by phagocytosis through inhibition of phagosomal acidification, and inhibition of phagolysosome formation. Fol- lowing uptake into macrophages, MTB resides within a modified lysosomal compartment, thereby avoiding the primary destructive mechanisms of the cell [2-4]. Modifica- tion of the lysosomal compartment provides an environment that facilitates replication and protection from antimicrobials [4-6].

Frequent failures of the treatment of tuberculosis (TB) are due to the insufficient dosing, incomplete treatment regimens caused by poor patient compliance, and latency profile of the bacilli [7, 8]. Initial treatment requires a two-step process; the first involves an “intensive phase” of a four-drug high dose cocktail containing the antibiotics rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB). The

“intensive phase” is limited to two-months to avoid the high toxicity issues found with this therapy regime. Following the “intensive phase” a longer “continuation phase”

166 occurs with RIF and INH and can last for up to two years [9-11]. This secondary treatment is necessary for the eradication of remaining bacilli that enter a dormant or latent phase during the early stages of infection. The lenghtly requirement of treatment highlights the unique pathogenesis of Mycobacterium, this remaining population of non- replicating “persistent state” is seen within the caseous core of lung granulomas [12].

These “persister” bacilli are genetically drug-susceptible but exhibit a phenotypic tolerance to currenty available first-line drugs [13, 14].

Although the incidence and mortality rates of TB have significantly declined within the last ten years, the advances in TB control remain hindered by the increase in reports of drug-resistance [14, 15]. Multidrug-resistant TB (MDR-TB), is defined as resistant to at least two antibiotics such as rifampicin and isoniazid [16, 17]. Extensively drug-resistant TB (XDR-TB) is a phenotypically adapted MDR which shows resistant to isoniazid and rifampin, plus fluoroquinolones and at least one of the injectable second- line drugs, such as amikacin [18, 19]. Furthermore, cases of totally drug-resistant tuberculosis (TDR-TB) have recently been reported in Asia, Africa, India and Europe.

TDR-TB is reported to be resistant to all available drug chemotherapy’s [20]. Most drug resistance is due to inadequate therapy regimes and pre-existing illnesses such as human immunodeficiency virus (HIV). There are two types of mycobacterial resistance; acquired resistance (i.e., environmental) or by infection with a drug-resistant strain (i.e., primary resistance) [21].

These intrinsic resistance mechanisms of MTB are thought to be the crucial link between a therapeutic cure and persistent infection [9, 22]. Because virtually all classes

167 of antibiotics require bacterial replication for their primary mode of action, the non-replicating state of the bacilli is believed to render MTB resistant to otherwise bactericidal antibiotics. This unique nature of the disease, specificity of location, and development of the unique granuloma structure contains and provides protection to persistent bacilli [23,

24], increasing the necessity for an antimicrobial treatment that provides continuous intracellular drug concentrations within these specialized sites. Therefore, there is a need to design novel antimicrobial delivery systems that effectively inhibit the growth and protect against multiple strains of M. tuberculosis whereby enhancing patient compliance by utilizing a condensed dose regimen.

This study demonstrates that the use of biodegradable polyanhydride nanoparticles can successfully encapsulate and release the small molecules rifampicin, isoniazid, pyrazinamide and ethambutol (RIPE) against M. tuberculosis. Polyanhydride’s have multiple benefits compared to other delivery systems [25]. These nanoparticles have shown sustained release profiles through bulk and surface erosion. The erosion profile provides a sustained release of cargo while maintaining the polymer reliability and function [26]. The use of polyanhydride nanoparticles comprised of 1,6-bis(p- carboxyphenoxy)hexane (CPH), 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), have been shown to be well suited for the delivery of small molecules and proteins through the pulmonary, subcutaneous and intraperitoneal administration. This delivery is further favorable based on the safety profile, mild inflammatory response, and their ability to enhance immunity [27, 28]. Additionally, polyanhydride nanoparticles have been shown to provide pathogen-mimicking capabilities that improve the internalization activity through functionalization and tailorable polymer combinations which have been

168 demonstrated to increase cellular uptake, particle persistence and accumulation within macrophages [25, 29, 30].

This work demonstrates a significant decrease in viable bacilli when tested within the human macrophage THP-1 in vitro. As well as, in vivo experiments will highlight the use of the nanoparticle-based delivery platform consisting of the RIPE drugs administered intraperitoneal to mice, reducing the prevalence of “persistor bacilli” when compared to soluble drugs. These studies provide a fundamental foundation for future work for the rational design of an anti-tuberculosis treatment based off of polyanhydride nanoparticles containing the RIPE drug cocktail.

Materials and methods

Nanoparticles

For the synthesis of the CPH and CPTEG monomers, the polyanhydride polymer,

20:80 CPTEG:CPH and the rifampicin, isoniazid, pyrazinamide and ethambutol-loaded polyanhydride nanoparticles, acetic acid, acetic anhydride, acetone, acetonitrile, chloroform, dimethyl formamide, ethyl ether, hexane, methylene chloride, pentane, petroleum ether, potassium carbonate, sodium hydroxide, sulfuric acid, and toluene were purchased from Fisher Scientific (Fairlawn, NJ).1-methyl-2-pyrrolidinone, hydroxybenzoic acid, N,N-dimethylacetamide, and tri-ethylene glycol were obtained from Sigma Aldrich (St. Louis, MO). The chemical 4-p-fluorobenzonitrile was purchased from Apollo Scientific (Cheshire, UK). For 1H NMR analysis deuterated chloroform and deuterated dimethyl sulfoxide were purchased Cambridge Isotope Laboratories (Andover,

MA). A fluorescent dye, Rhodamine B, was purchased from Sigma Life Science (St.

169

Louis, MO). Rifampicin, isoniazid, pyrazinamide, and ethambutol were also purchased from Sigma Life Science (St. Louis, MO).

Polymer Synthesis

Synthesis of the monomers used, 1,6-bis(p-carboxyphenoxyhexane) (CPH) and

1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) was performed as described elsewhere [31]. 20:80 molar copolymers of CPTEG and CPH were synthesized through melt condensation polymerization, as previously described in detail [26, 32, 33]. The molecular weight was confirmed using 1H NMR.

Nanoparticle Fabrication

Rifampicin, isoniazid, pyrazinamide and ethambutol-loaded nanoparticles of

28:80 CPTEG:CPH were fabricated through solid/oil/oil nanoprecipitation, as previously described [34-36]. Drug combinations Rifampicin and pyrazinamide, each at 5% (w/w), were added to the polymer. Drug combinations, isoniazid, and ethambutol each at 5%

(w/w) were added to the polymer. The solid drug and polymer mixture was then dissolved in methylene chloride at a concentration of 20 mg/mL followed by rapid precipitation into the antisolvent, pentane. The polyanhydride nanoparticles were then collected using vacuum filtration. The drug-loaded particles were characterized for size and morphology by scanning electron microscopy (SEM, FEI Quanta SEM, Hillsboro, OR).

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Drug release kinetics

Rifampicin, pyrazinamide, and isoniazid were quantified by RP-HPLC. RIF and

PZA release samples in PBS were separated using a Phenomenex Kinetex 2.6-micron

C18 100Å 100x4.6mm column and a mobile phase consisting of a 73:20:7 volume ratio of water: acetonitrile : methanol. The flow rate was set to 0.4 mL/min, and rifampicin and pyrazinamide eluted at 4.8 min and 2.8 min, respectively. Rifampicin and pyrazinamide were detected by absorbance at 262 nm and 326 nm. Rifampicin and pyrazinamide base extraction samples in 40 mM NaOH were separated using a Zorbax SB-C8 5-micron

4.6x150mm column. A mobile phase gradient was used, ramping from 83:2:15 to

55:30:15 water : acetonitrile : tetrahydrofuran. The flow rate was 1 mL/min, and rifampicin and pyrazinamide eluted at 14.1 min and 1.4 min, respectively. RIF and PZA were detected by absorbance at 262 nm and 333 nm, respectively. Isoniazid release samples in

PBS were separated using a Phenomenex Kinetex 2.6-micron C18 100Å 100x4.6mm column and a mobile phase consisting of a 95:5 volume ratio of water : acetonitrile. The flow rate was set to 0.4 mL/min, and isoniazid eluted at 4.3 min. IZD base extraction samples in 40 mM NaOH were separated using a Zorbax Eclipse XDB-C8 5-micron

4.6x150mm column. A mobile phase gradient was used, ramping from 98:2 to 90:10 water : acetonitrile. The flow rate was set to1 mL/min, and isoniazid eluted at 1.1 min.

Isoniazid PBS release and base extraction samples were detected. Stocks of each drug in

PBS and 40 mM NaOH were prepared. Dilution series of these stocks in PBS and NaOH were used to quantify drug concentration in each sample by absorbance at 260 nm.

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Mycobacteria and culture conditions.

M. tuberculosis strains (H37Rv, HN563 and NR49360) were propagated at 37°C either in Middlebrook 7H9 broth (Difco) containing 10% oleic acid-albumin-dextrose- catalase (Difco) and 0.05% Tween 80 (Sigma) or on Middlebrook 7H10 or 7H11 agar containing 10% oleic acid-albumin-dextrose-catalase. Cycloheximide (50 μg ml−1), amphotericin B (50μg ml -1) and carbenicillin (15 μg ml-1) was included in the media to prevent fungal and as a selective agent. Frozen stocks were prepared by growing bacteria in 7H9 broth to mid-log phase (optical density at 600 nm [OD600], ∼0.5), collecting cells by centrifugation, washing once with phosphate-buffered saline containing 0.05% Tween

80 (PBST), adding glycerol to 15%, and storing in aliquots at −80°C. Bacterial stocks were titered by plating PBST-diluted bacterial suspensions on 7H10 agar and scoring colonies after 3 to 4 weeks at 37°C.

THP-1 cell cultures

The human monocytic cell line THP-1 was cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS). Suspension cultures of THP-1 cells were maintained at a cell density of 2 × 106 cells/ml of cells in culture medium and split two times per week. THP-

1 cells growing in suspension were harvested at a density of 1 × 106 cells/ml, and hemocytometer and trypan blue dye assessed cell viability.

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Table 7-1 Level of DNA relatedness among strains of Mycobacterium species

Level of DNA relatedness among strains of mycobacterial species[37, 38]

Level of relatedness % to guanine and cytosine:

Species

M. tuberculosis H37Rv M. tuberculosis HN563 M. tuberculosis NR 49360 M. paratuberculosis M. marinum M. tuberculosis ____ N/A 99 <.1 86 H37Rv M. tuberculosis N/A* N/A* N/A* N/A* N/A* HN563 M. tuberculosis NR 99 N/A* ____ 45 85.4 49360 M. paratuberculosis <.1 N/A* <.1 ____ 45 M. marinum 86 N/A* 85.4 45 ____

N/A* The complete genome of M. tuberculosis, strain HN563 is currently being sequenced. ---- Non-applicable

Cells were resuspended in fresh medium supplemented with 5 nM phorbol myristic acid (PMA) to terminally differentiate the cells into adherent monocytes.

Suspensions were adjusted to a concentration of 0.5 x105 cells/ml in warm supplemented

RPMI, seeded in 96-well plates adding 200 [micro]l of suspension per well, and cells were allowed to adhere and differentiate, at 37°C in 5% CO2 atmosphere for 24 h.

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Table 7-2 Mycobacterial drug susceptibility

Mycobacterial drug susceptibility MIC (mg/L)[39]

Bacterial Species

Isoniazid mg/L Serum Pyrazinamide mg/L Serum Rifampicin mg/L Serum Ethambutol mg/L Serum

Mycobacterium 0.05-0.2 5-10 20 40-50 0.5 10 1-5 2-5 tuberculosis wild-type

Broth microdilution method.

Antibiotic testing against M. tuberculosis HN563 and NR49360 were determined in 7H9 broth by the standard microdilution method. Briefly, 0.1-ml of a 0.1 mg ml-1 dilution of RIPE drugs were added to a 96-well plate. Control wells included medium and culture controls. One hundred microliters of culture (at 0.5 x 106 CFU/ml) was added to all the wells except the medium control wells. The plates were incubated at 37°C for up to 7 days. Following each incubation time intervals, twenty microlitres of each bacterial suspension were serially diluted in PBS and aliquots of triplicate were plated onto Mid- dlebrook 7H10 or 7H11 media. The plates were sealed and incubated for 18–26 days at

37°C. Experiments were performed in duplicate and the standard deviation is depicted on graphs unless otherwise stated.

Intracellular viability and replication of the M. tuberculosis in THP-1 cells.

THP-1 cells used for evaluation of the intracellular survival of M. tuberculosis strains were treated similarly as previously described in 96-well flat-bottom tissue culture

174 plates. Bacterial suspensions were prepared and generated by scraping 14 d cultures of the M. tuberculosis strains grown on Middlebrook 7H10 agar into screw-cap microfuge tubes containing 7H9 media and incubated for 96 h. Pellets of bacteria were resuspended through mechanical disruption and numbers of bacteria present in the suspensions were determined by optical density at 600 nm measurements[40]. These suspensions were used to generate dilute M. tuberculosis using complete RPMI 1640 medium whereby the bacterial density was adjusted to desired levels to account for variations in the numbers of target monocytes. Suspensions of bacteria were added to monocyte monolayers at a multiplicity of infection (bacteria/monocyte ratio) of 10:1 for M. tuberculosis H37Rv,

NR49360, and HN563. Tissue culture plates were gently agitated by hand and then centrifuged at 4°C for 10 min at 270 x g. Moreover, transferred to an incubator for 90 min

37°C with 5% CO2 to allow for phagocytosis of bacteria. Monolayers were washed three times with PBS to remove any remaining nonadherent bacteria. Fresh RPMI-1640 with

10 ug/ml gentamicin complete media was added following the last washing. The viability of intracellular Mycobacterium was determined by lysing monocytes with 0.1% deoxycholate, diluting suspensions in PBS, and plating aliquots in triplicate on Middlebrook

7H10 and 7H11 medium. The plates were sealed and incubated for 18–26 days at 37°C.

Total cfu/ml of bacterial survival at 24, 48 and 72 h were calculated based on the number of internalized bacteria detected at 1.5 h postinfection which represents 100% of internalized bacteria. Experiments were performed in duplicate and the standard deviation is depicted on graphs unless otherwise stated.

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Acute Mycobacterium infection in animals

Male C3H/HeNCrl mice (6–8 weeks old) was purchased from Charles River

(Wilmington, MA, USA). All animals were housed in a biosafety level 3 facility and procedures related to mice followed protocols approved by the Institutional Animal Care and

Use Committee at Iowa State University. This species of mice was chosen for the ability to model both the systemic/acute phase of tuberculosis ( < 2 weeks post i.p. infection) and the chronic/granulomatous phase infection ( > 6 weeks post i.p. infection)[41]. Paren- teral administration of infectious doses were through intraperitoneal injection of thawed aliquots that were prepared by dilution to ∼0.4 104 CFU ml−1 in PBST, vortexed (15 s) to disperse clumps, and injection of 0.5 ml (∼2000 CFU)

Animal chemotherapy

Chemotherapy was initiated 14 days after intraperitoneal infection. Mice received intraperitoneal administration the RIPE drugs as either soluble or encapsulated into 20:80

CPTEG:CPH. Dosing was once daily for seven consecutive days with 12 mice per treatment group. The soluble RIPE treatment group received 0.15 mg/kg soluble rifampicin, isoniazid, pyrazinamide, and ethambutol in 500 μl PBS. NP-RIPE group received five mg of encapsulated 20:80 CPTEG:CPH nanoparticle containing a total drug concentration of

0.15 mg/kg rifampicin, isoniazid, pyrazinamide, and ethambutol in 500 μl PBS. During the study period, mice were weighed weekly to monitor the mean animal body weights.

Bacterial burden present within target tissues of spleen, liver, and lungs were quantified by harvesting tissues 2 days following the week of treatment. Mice were

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euthanized by CO2 overdose, right lungs, left liver lobe, and whole spleens were aseptically removed and these organs were transferred into 2 ml tubes each containing 1 ml sterile distilled water and 0.2 mm diameter stainless steel beads for homogenization for 30 seconds of 30,000 rpm with a tissue homogenizer (1-3 times) (BioSpec Products

Inc., Bartlesville, OK) CFU counts of the organs were performed using the diluted homogenate in PBS, and plating of aliquots in triplicate on Middlebrook 7H10 and 7H11 plates (Becton Dickinson, Sparks, MD, USA) for cfu enumeration, as previously described. Data are derived from 12 mice per treatment group and 8 mice for the untreated controls. The remaining left lung and liver from each animal were fixed in 10% buffered formaldehyde for histopathology.

The screening of an independent, drug-sensitive persister population

Presence of a persister population was tested in a 96-well plate adding 100 μl of tissue homogenate suspension (liver, spleen, lung) per well. Briefly, 0.1-ml of a 0.01 mg ml-1 dilution of rifampicin drug was suspended into 7H9 culture broth and added to tissue homogenates and incubated at 37C 5% CO2 for 96 h. Following incubation, twenty microlitres of each bacterial suspension were serially diluted in PBS and aliquots of triplicate were plated onto Middlebrook 7H10 or 7H11 media. The plates were sealed and incubated for 18–26 days at 37°C.

Statistical analysis

The statistical software GraphPad Prism (v.7.04) was used to analyze the data.

Two-way ANOVA and Student t-test were used to determine statistical significance

177 among treatments and p< 0.05 was considered significant. Standard deviation is depicted unless otherwise stated.

Results

Mycobacterium tuberculosis strains H37Rv (WT-TB), HN563 (MDR-TB) and

NR49360 (XDR-TB) was tested for (Figure 7-1, 2, and 3) susceptibility and sensitivity to the four drug cocktail rifampicin, pyrazinamide, isoniazid, and ethambutol (RIPE) either as soluble or encapsulated into 20:80 CPTEG:CPH nanoparticle. Mycobacterium cultures were incubated with a single dose of decreasing amounts of drug conducted over seven days with subsequent plating of bacterial enumerations. RIPE concentrations above 0.32 ug/ml were inhibitory for both soluble and NP treated cultures. The XDR-TB exhibited increased sensitivity to the nanoparticles at the highest and mid-range (64 and 10 μg/ml) concentration. NP encapsulation showed 2.01 log CFU (± 0.83) killing activity over soluble at the 64 μg/ml. The ability of the XDR-TB strain to survive within the highest concentration tested of the 4 drug RIPE cocktail demonstrates the “extensive” nature of resistance of this isolate and is consistent with published reports for this and other XDR strains.

The MDR-TB (Figure 7-2) was also susceptible to the nanoparticle and soluble drugs for all concentrations tested. However, the 20:80 CPTEG:CPH nanoparticle group provided the highest inhibition of MDR-TB. An almost 200 fold decrease in the drug was observed to achieve an 8.32 log CFU (± 0.83) difference within the bacterial population.

The H37Rv strain showed similar sensitivity to the drug cocktail as was seen in the

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MDR-TB. These results provide the opportunity to further examine the intracellular events of bacterial infection and effectiveness of the drug cocktail.

Figure 7-1 Antimicrobial activity of NP-RIPE against XDR-TB M. tuberculosis NR49360 Enu- meration of MTB CFU from the 48hr treatment of broth cultures with decreasing concentrations of soluble and NP-RIPE formulations. (Student’s T-test p values * < 0.05, ** < 0.01).

To examine the intracellular fate of Mycobacteria three strains H37Rv, MDR-TB, and XDR-TB we (Figure 7-4) tested at an MOI of 10:1 in the human macrophage cell line THP-1. Infectious bacteria where allowed 24 h to reach their ecological niche before a single application of the previously mentioned drug cocktail was applied and monitored over 48 h. For the H37RV (Figure 7-3) strain at the at the 10 μg/ml concentration the NPs decreased the survivability of mycobacterium nearly 5.23 log CFU (± 0.59) and the soluble drug group also provided a ⁓ 4.5 log CFU (± 0.54) reduction in the bacterial population. The MDR-TB showed susceptibility to the drug cocktail.

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Figure 7-2 The killing of M. tuberculosis MDR-TB enhanced by using nanoparticles loaded with rifampicin, pyrazinamide, isoniazid and ethambutol in broth. Colony forming units/ml were enumerated at 48 hrs post-infection and compared with the CFU/ml values for soluble treated bacteria. (Student’s T-test p values * < 0.05, ** < 0.01).

Intracellular MDR-TB (B) was more sensitive to RIPE encapsulated in NP group than soluble reflected in reducing the bacterial population an averaged 4.90 log

CFU ( ± 0.61) and nearly a 3.04 log CFU (± 0.56 ). There were significant differences found between the total log CFU when we directly compared the nanoparticle and soluble group (p <0.05). The XDR-TB showed limited sensitivity to the drugs, which was to be expected due to the natural resistance mechanisms. Although, the nanoparticle group did provide a 1.64 log CFU (± 0.13) reduction in the bacterial population and had insufficient

180 variability. Whereas, the soluble group provided a 1.01 log CFU (± 0.67) bacterial population reduction but had considerable variability between each replicate.

Figure 7-3 Killing of M. tuberculosis H37Rv enhanced by using nanoparticles loaded with rifampicin, pyrazinamide, isoniazid and ethambutol in broth. CFU units/ml were enumerated at 48 hrs post-infection and compared with the CFU/ml values for soluble treated bacteria. (Student’s T- test p values * < 0.05, ** < 0.01). The in vivo activity of the four drug cocktail as either soluble or encapsulated into

20:80 CPTEG:CPH nanoparticle using the C3H mouse model. An early acute phase My- cobacterium infection model was employed by infecting mice with one the following M. tuberculosis strains H37Rv, MDR-TB, and XDR-TB. Mice were infected intraperitoneal, and treatment began fourteen days post infection with daily 0.15mg/kg doses of drug cocktail given over seven consecutive days. Tissues from the spleen, liver, and luge were collected for either homogenization or histopathology and mean change is weight was

181 also accessed. The total bacterial population was lower than expected for all excised tissues, but due to the constraints of the experiment, this was to be expected.

The Mycobacterium H37Rv (Figure 7-5) strain, was actively establishing an acute infection within the tissues collected. Inhibition of Mycobacterium was seen in the spleen and lung in the nanoparticle (P < 0.05) treated mice. The nanoparticle formulation lowered the 0.815 log CFU (± 1.20) for the spleen and 2.56 log CFU (± 1.31) for the lung, respectively. The solubly treated groups did not reduce the total CFU within any tissues sampled, and overall development of a resister bacterial population was not evident.

The MDR-TB strain was actively establishing infection in the liver, spleen and lung (Figure 7-6). The soluble group and the nanoparticle group showed an increased in bactericidal (p<0.05) activity in the spleen. Soluble presented a 1.21 log CFU (±1.24) and

NP 1.18 log CFU (±1.20) reduction in the bacterial population compared to untreated.

MDR-TB did not appear affected by soluble or encapsulated drug in the liver or lung tissue. The development of a statistically significant (P<0.05) set of persister bacteria emerge within the spleen and lungs and no bacteria were recovered from the liver. observed considerable variability in the total recovered bacteria between the tissues and observed an increase in the bacterial burden was found in the spleen (p< 0.05) for the nanoparticle group 0.31 log CFU (± 0.35) compared to the untreated.

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Figure 7-4 Continued on next page.

183

Figure 7-4 Intracellular viability of Mycobacterium tuberculosis in the Human macrophage THP- 1 (continued from previous page). Three strains of Mycobacteria where infected at an MOI of 10:1 and monitored over 24 h. (A) WT-TB H37Rv, (B) MDR-TB HN563, and (C) XDR-TB NR49360. Before treatment of the drug cocktail, RIPE was applied as a single dose for 48 h. The cell lysate was serially diluted and was plated onto 7H10 agar plates for CFU bacterial survival. Student t-test was performed for significance between the nanoparticle and soluble group (<0.01).

The Mycobacterium XDR-TB strain (Figure 7-7) was active in the spleen, liver, and lung for both the soluble and nanoparticle treatment groups. Unlike the H37Rv and

MDR-TB, the XDR-TB was the most actively established of the three strains. The XDR-

TB was most active in the liver and lung (P < 0.05), the average CFU recovered for the nanoparticle group for the liver was 4.35 log CFU (±0.69) and lung 2.80 log CFU (±

1.43). The soluble group also had minimal effects against XDR-TB, CFUs recovered from the spleen 4.63 log CFU (± 0.47), liver 5.05 log CFU (± 1.77), and lung 2.99 log

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CFU (± 1.53). Development of a persister population was evident with the solublly treated mice having the largest population of bacteria was recovered from the spleen 3.37 log CFU (± 1.76) and lung of 3.65 log CFU (± 1.71), this is in sharp contrasts to the nanoparticle and untreated. We did not recover any viable persister bacterial from the liver for all groups tested.

Figure 7-5 The use of the drugs against H37Rv in vivo. Use of the four drugs RIPE following a acute infection in C3H mice with Mycobacterium tuberculosis H37Rv. The graph is a representation of dose-response and development of a persister bacterial population in mice. Stu- dent t-test was performed for significance between the nanoparticle and soluble group (<0.05).

185

Figure 7-6 The use of drugs against HN563 (MDR) in vivo. Development of an acute infection in C3H mice with Mycobacterium tuberculosis HN563 (MDR). The graph is a representation of dose-response and development of a persister bacterial population in mice. Student t-test was performed for significance between the nanoparticle and soluble group (<0.05).

The C3H mice’s weights (Figure 7-8) over the course of the experiment were monitored and did found no ill effect was found for all groups. Results from histopathology (N=3) did not show any evidence of a lesions in the liver, spleen or

186 granuloma formation in the lung. Further, acid-fast histochemical stains for organisms were negative.

Figure 7-7 The use of the drugs against XDR-TB in vivo. Mycobacterium tuberculosis NR49360 (XDR) presented as an acute infection in C3H mice over a 22 d infection. The graph is a representation of dose-response and development of a persister bacterial population in mice. Student t- test was performed for significance between the nanoparticle and soluble group (<0.05).

Using an HPLC assay as described in the methods section, the release kinetics of rifampicin, pyrazinamide, and isoniazid from nanoparticles was quantified, as shown in

Figure 7-9. The 20:80 CPTEG:CPH nanoparticle formulation for the three drugs shown-

187 indicate an initial burst release profile, followed by sustained release is shown in the isoniazid nanoparticles. It was also observed that there was a much larger burst release of pyrazinamide as compared to that of rifampicin within the first 24 h.

Figure 7-8 C3H mice weights where collected once every seven days over twenty-two days. (A) WT-TB , (B) MDR-TB, and (C) XDR-TB. There were no significant differences found between the untreated and those that were either treated with soluble or encapsulated rifampicin, isoniazid, pyrazinamide or ethambutol. Significance was determined using a student t-test.

Encapsulation efficiency was also quantified, and revealed in the dual encapsulation of pyrazinamide with rifampicin the total drug encapsulated was significantly less

188 than expected. The pyrazinamide encapsulated at 60 ± 0.97 % and rifampicin 80 ± 0.87

%. The nanoparticles containing the isoniazid and ethambutol showed a 58.08 ± 0.52 % encapsulation efficiency for isoniazid, respectively. Ethambutol was not quantified.

Figure 7-9 The in vitro release profiles of pyrazinamide, rifampicin, and isoniazid. Total fraction release of drug over time quantified by HPLC. Total released drug over 48 h, 20:80 CPTEG:CPH nanoparticles. Dash line indicated 100% drug release.

Nanoparticles used in the in vivo and HPLC assay where evaluated against M. marinum towards bacterial susceptibility of the released free four drug cocktail drugs from 20:80 CPTEG:CPH nanoparticles. Released drugs were tested in comparison to a known volume and concentration of soluble drug cocktail and applied to a bacterial population and monitored over time (Figure 7-10). The solubly treated group of 10 μg did not demonstrate an inhibitory effect over the treatment duration. The released drugs from the

189

24 h time point also showed an initial burst of drugs was released as seen in the HPLC assay. The majority of the total drug was released from the nanoparticles within the first 72 h and provided minimal effects against M. marinum at later time points (data not shown).

Figure 7-10 Inhibitory activity of the released drug against M. marinum. 20:80 CPTEG:CPH NPs used in the in vitro assay were examined for total drug encapsulation and released drug over time. Supernatants collected at specified time points from the NPs were applied at equivalent volumes relative to the solubly treated groups and monitored over nine days. At determined time points, bacterial enumerations were plated onto select media and monitored for CFU/ml. Student t-test was performed for significance between the nanoparticle and soluble group (<0.05)

Discussion

This current work demonstrates the importance of rationally designing delivery vehicles for drug optimization. Because of the known differences in drug sensitivity among Mycobacterium, the need for the development of an innovative drug delivery platform that can overcome these factors must be considered for treatment of the chronic phase. The encapsulation of the drugs rifampicin, isoniazid, pyrazinamide and

190 ethambutol into polyanhydride nanoparticles provided benefits over the use of soluble drugs including; colocalization, increased drug efficacy, and dose sparing.

In the in vitro Mycobacterium broth assay, polyanhydride nanoparticles improved upon the antimicrobial activity of soluble drugs against MDR-TB and XDR-TB. We found that an increased concentration range of drug was minimally active against the

MDR-TB and XDR-TB without encapsulation. The soluble inhibition breakpoint for the

MDR-TB was found to be <64μg/ml which is higher than previously published for this strain. The most significant effect of the drug cocktail was seen with the use of the nanoparticles as a delivery vehicle. A susceptible breakpoint of 1.6μg/ml, and resistance was not evident at the lowest concentration tested of 0.032 μg/ml. The XDR-TB the inhibition breakpoint for the soluble and nanoparticle groups was <64 μg/ml, with resistance observed at > 1.6μg/ml. There where minor differenced in the soluble groups total CFU/ ml for the MDR-TB and XDR-TB when comparing the two assays to each other.

The Human macrophages THP-1 was infected with Mycobacterium at an MOI of

10:1. The decrease in the bacterial population suggests colocalization of the nanoparticle occurred with both the MDR-TB and XDR-TB. We previously have shown that the highly virulent intracellular pathogen Brucella co-localizes with the nanoparticles within infected cells and this observation correlates with increased intracellular bacterial killing

[29, 30, 42]. Similar experiments assessing changes in MTB intracellular survival in the presence of the NP-RIPE drug cocktail on MTB survival were described in this work whereby a single 10 μg/ml dose of either soluble or encapsulated RIPE was given to

MTB infected human macrophages. While NP-RIPE and soluble RIPE treatment equally

191 reduced intracellular viability in WT MTB strain H37Rv, NP-RIPE reduced bacterial counts to an amount significantly lower than soluble demonstrating the increased efficacy of NP delivery of the four drug cocktail. It is of note that soluble groups in this intracellular infection study consistently produced higher amounts of variability in CFU recovered than either NP-RIPE or untreated groups. Within the treatment group, increases in the variability (1-2 log) for the soluble group whereas in the nanoparticle group this was not observed.

Microbial drug persistence is a widespread phenomenon in which a sub-population of microorganisms can survive antimicrobial treatment without the development of extrinsic resistance. This small sub-population of Mycobacterium that survives the lethal effects of the antibiotic drug is referred to as “persister” [43]. Persistence is distinct from resistance in that, unlike resistant mutants, persister populations do not undergo active replication in the presence of the antibiotics (“stressor”), and population growth resumes only once the drug has been removed [44]. This stress response is a mechanism by which

Mycobacterium is inherently known for, and provides evidence for the development of a latent infection.

The presence of a persister bacterial population within the MDR-TB and XDR-

TB treatment groups was further exacerbated after treatment with both the soluble and

NPs. It appears that the primary treatment with NP provided a large enough stress induced persistence response by which Mycobacterium entered into a dormancy state earlier than in the untreated groups. This may suggest that the activity of the NPs in the delivery of antibiotics provided a large enough stress response to arrest the cellular growth prematurely whereby inhibiting the action of the antibiotics. Additional studies to

192 confirm this will need to occur. The lack of a viable hepatic TB population as well as granuloma formation within the lungs. Studies have shown that individuals can develop granulomas of the lung and hepatic lesions within fourteen days of exposure and those that tested positive for pulmonary TB also test positive for hepatic TB 50-80% of the time [45, 46]. We can only attribute this lack of granuloma and hepatic lesion development primarily to the early acute phase.

Conclusion

We demonstrated the use of polyanhydride nanoparticle as an alternative drug delivery platform for the treatment of WT-TB, MDR-TB, and XDR-TB. This is the first reported multi-strain M. tuberculosis in vivo study conducted with the use of polyanhydride nanoparticles and the four drug cocktail rifampicin, pyrazinamide, ethambutol, and isoniazid. Our in vitro studies provided an insight into the benefits of encapsulating rifampicin, isoniazid, pyrazinamide, and ethambutol. We observed the use of encapsulated antituburculars showed an increased drug efficacy and dose sparing when used against WT-TB, MDR-TB, and XDR-TB in vitro. Despite the reduced sustained drug release profile and reduced encapsulation efficacy, the utilization of amphiphilic polyanhydride nanoparticles still showed benefits in vivo. Together, this data exemplify the physiochemical properties of polyanhydride nanoparticles against Mycobacterium.

This work has provided the foundational framework for future studies focused on the interactions between Mycobacterium and use of polyanhydride nanoparticle.

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References

1. Johnson, M.M. and J.A. Odell, Nontuberculous mycobacterial pulmonary infections. Journal of Thoracic Disease, 2014. 6(3): p. 210-220.

2. Johansson, J., et al., Phagocyte interactions with Mycobacterium tuberculosis — Simultaneous analysis of phagocytosis, phagosome maturation and intracellular replication by imaging flow cytometry. Journal of Immunological Methods, 2015. 427: p. 73-84.

3. Koster, S., et al., Mycobacterium tuberculosis is protected from NADPH oxidase and LC3-associated phagocytosis by the LCP protein CpsA.(MICROBIOLOGY)(Report). Proceedings of the National Academy of Sciences of the United States, 2017. 114(41): p. E8711.

4. Weiss, G. and U.E. Schaible, Macrophage defense mechanisms against intracellular bacteria. Immunological Reviews, 2015. 264(1): p. 182-203.

5. Srivastava, S., et al., Susceptibility Testing of Antibiotics That Degrade Faster than the Doubling Time of Slow-Growing Mycobacteria: Ertapenem Sterilizing Effect versus Mycobacterium tuberculosis. Antimicrobial agents and chemotherapy, 2016. 60(5): p. 3193.

6. Ouadrhiri, Y., et al., Mechanism of the Intracellular Killing and Modulation of Antibiotic Susceptibility of Listeria monocytogenes in THP-1 Macrophages Activated by Gamma Interferon. Antimicrobial Agents and Chemotherapy, 1999. 43(5): p. 1242-1251.

7. Hoagland, D.T., et al., New agents for the treatment of drug-resistant Mycobacterium tuberculosis. Advanced Drug Delivery Reviews, 2016. 102: p. 55-72.

8. Izadi, N., et al., Co-infection of long-standing extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) and non-tuberculosis mycobacteria: A case report. Respiratory Medicine Case Reports, 2015. 15: p. 12-13.

9. Prat, C. and A. Lacoma, Bacteria in the respiratory tract-how to treat? Or do not treat? Int J Infect Dis, 2016. 51: p. 113-122.

10. Connolly, L.E., P.H. Edelstein, and L. Ramakrishnan, Why Is Long-Term Therapy Required to Cure Tuberculosis? PLoS Medicine, 2007. 4(3): p. e120.

11. Organization, W.H. Global tuberculosis report 2017. 2017 [cited 2017 December 01]; Available from: http://www.who.int/tb/publications/global_report/en/.

194

12. Vandiviere, H.M., et al., The treated pulmonary lesion and its tubercle bacillus. II. The death and resurrection. Am J Med Sci, 1956. 232(1): p. 30-7; passim.

13. Ahmad, Z., et al., Mycobacterium tuberculosis Effectiveness of tuberculosis chemotherapy correlates with resistance to Mycobacterium tuberculosis infection in animal models. Journal of Antimicrobial Chemotherapy, 2011. 66(7): p. 1560- 1566.

14. Smith, T., K.A. Wolff, and L. Nguyen, Molecular Biology of Drug Resistance in Mycobacterium tuberculosis. Current topics in microbiology and immunology, 2013. 374: p. 53-80.

15. Jing, W., et al., Rifabutin Resistance Associated with Double Mutations in rpoB Gene in Mycobacterium tuberculosis Isolates. Front Microbiol, 2017. 8: p. 1768.

16. Berrada, Z.L., et al., Rifabutin and rifampin resistance levels and associated rpoB mutations in clinical isolates of Mycobacterium tuberculosis complex. Diagn Microbiol Infect Dis, 2016. 85(2): p. 177-81.

17. Jamieson, F., et al., Profiling of rpoB Mutations and MICs for Rifampin and Rifabutin in Mycobacterium tuberculosis. Journal of Clinical Microbiology, 2014. 52(6): p. 2157.

18. Saravanan, M., et al., Review on emergence of drug-resistant tuberculosis (MDR & XDR-TB) and its molecular diagnosis in Ethiopia. Microb Pathog, 2018. 117: p. 237-242.

19. Yinnon, A.M., et al., Emergence of drug-resistant tuberculosis in Jerusalem: ten- year retrospective review. Isr J Med Sci, 1997. 33(11): p. 728-33.

20. Klopper, M., et al., Emergence and Spread of Extensively and Totally Drug- Resistant Tuberculosis, South Africa. Emerging Infectious Diseases, 2013. 19(3): p. 449-455.

21. Shah, N.S., et al., Worldwide Emergence of Extensively Drug-resistant Tuberculosis. Emerging Infectious Disease journal, 2007. 13(3): p. 380.

22. Nguyen, L., Antibiotic resistance mechanisms in M. tuberculosis: an update. Archives of toxicology, 2016. 90(7): p. 1585-1604.

23. Agrawal, N., et al., Dissecting host factors that regulate the early stages of tuberculosis infection. Tuberculosis, 2016. 100: p. 102-113.

24. O'Garra, A., et al., The Immune Response in Tuberculosis. Annual Review of Immunology, 2013. 31(1): p. 475-527.

195

25. Ross, K.A., et al., Nano-enabled delivery of diverse payloads across complex biological barriers. J Control Release, 2015. 219: p. 548-559.

26. Torres, M.P., et al., Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. Journal of biomedical materials research. Part A, 2006. 76(1): p. 102.

27. Ross, K.A., et al., Lung deposition and cellular uptake behavior of pathogen- mimicking nanovaccines in the first 48 hours. Adv Healthc Mater, 2014. 3(7): p. 1071-7.

28. Vela Ramirez, J.E., et al., Polyanhydride Nanovaccines Induce Germinal Center B Cell Formation and Sustained Serum Antibody Responses. J Biomed Nanotechnol, 2016. 12(6): p. 1303-11.

29. Goodman, J.T., et al., Nanoparticle Chemistry and Functionalization Differentially Regulates Dendritic Cell-Nanoparticle Interactions and Triggers Dendritic Cell Maturation.(Report). Particle & Particle Systems Characterization, 2014. 31(12): p. 1269.

30. Phanse, Y., et al., Functionalization promotes pathogen-mimicking characteristics of polyanhydride nanoparticle adjuvants. J Biomed Mater Res A, 2017. 105(10): p. 2762-2771.

31. Torres, M.P., et al., Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. J Biomed Mater Res A, 2006. 76(1): p. 102-10. 32. Domb, A.J., et al. Polyanhydrides: Synthesis and characterization. 1993. Berlin, Heidelberg: Springer Berlin Heidelberg.

33. Kipper, M.J., et al., Design of an injectable system based on bioerodible polyanhydride microspheres for sustained drug delivery. Biomaterials, 2002. 23(22): p. 4405-12.

34. Haughney, S.L., et al., Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles. Acta Biomater, 2013. 9(9): p. 8262-71.

35. Ulery, B.D., et al., Design of a protective single-dose intranasal nanoparticle- based vaccine platform for respiratory infectious diseases. PLoS One, 2011. 6(3): p. e17642.

36. Huntimer, L., et al., Evaluation of biocompatibility and administration site reactogenicity of polyanhydride-particle-based platform for vaccine delivery. Adv Healthc Mater, 2013. 2(2): p. 369-78.

196

37. Eisenach, K.D., J.T. Crawford, and J.H. Bates, Genetic Relatedness among Strains of the Mycobacterium tuberculosis Complex. American Review of Respiratory Disease, 1986. 133(6): p. 1065-1068.

38. Stinear, T.P., et al., Comparative genetic analysis of Mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent divergence. Journal of bacteriology, 2000. 182(22): p. 6322-6330.

39. Böttger, E.C., The ins and outs of Mycobacterium tuberculosis drug susceptibility testing. Clinical Microbiology and Infection, 2011. 17(8): p. 1128-1134.

40. Peñuelas-Urquides, K., et al., Measuring of Mycobacterium tuberculosis growth. A correlation of the optical measurements with colony forming units. Brazilian Journal of Microbiology, 2013. 44(1): p. 287-289.

41. Kramnik, I. and G. Beamer, Mouse models of human TB pathology: roles in the analysis of necrosis and the development of host-directed therapies. Seminars in Immunopathology, 2016. 38: p. 221-237.

42. Ulery, B.D., et al., Polymer chemistry influences monocytic uptake of polyanhydride nanospheres. Pharm Res, 2009. 26(3): p. 683-90.

43. McCune, R.M., R. Tompsett, and W. McDermott, THE FATE OF MYCOBACTERIUM TUBERCULOSIS IN MOUSE TISSUES AS DETERMINED BY THE MICROBIAL ENUMERATION TECHNIQUE : II. THE CONVERSION OF TUBERCULOUS INFECTION TO THE LATENT STATE BY THE ADMINISTRATION OF PYRAZINAMIDE AND A COMPANION DRUG. THE JOURNAL OF EXPERIMENTAL MEDICINE, 1956. 104(5): P. 763-802.

44. Cohen, N.R., M.A. Lobritz, and J.J. Collins, Microbial persistence and the road to drug resistance. Cell host & microbe, 2013. 13(6): p. 632-642.

45. Hussain, W., et al., Fulminant hepatic failure caused by tuberculosis. Gut, 1995. 36(5): p. 792-794.

46. Singh, S., et al., Primary hepatic tuberculosis: a rare but fatal clinical entity if undiagnosed. Asian Pac J Trop Med, 2012. 5(6): p. 498-9.

CHAPTER 8 . CONCLUSIONS AND FUTURE WORK

The long-term goals of this research are to design an innovative nanotechnology delivery platform that can be utilized in multiple applications. The many challenging aspects of parasitic and intracellular pathogen treatment include the unique multi-stages of disease and location of the pathogen within a host must all be considered in developing novel therapies. The effectiveness of polyanhydride nanoparticles was validated through this research by improving the delivery of small hydrophilic/hydrophobic molecules and targeting of infected tissues. All these benefits highlight the use of polyanhydrides as a delivery platform. Beneficial aspects of drug delivery observed in this work include increasing drug efficacy, tailoring drug release profiles, and the ability to encapsulate small molecules. In this regard, examining the use of altered polymer combinations, surface functionalization and size could be optimized to fit these specific applications for future studies.

In chapter two we investigated the interaction of polyanhydride nanoparticle as a drug delivery platform for the antibiotics ivermectin and doxycycline against one of the causative agents of lymphatic filariasis, B. malayi. The co-delivery of doxycycline and ivermectin in polyanhydrides improved killing of adult parasites with up to 4,000 fold less drug compared to standard, soluble drug administration. Examination of worms by confocal microscopy revealed the rapid penetration of polyanhydride nanoparticles inside the worm suggesting rapid translocation across the cuticle. The delivery of micromolar concentrations of ivermectin and doxycycline reduced the shedding and viability of microfilaria. These results support the hypothesis; enhanced killing of the macrofilaria 198 results from the nanoparticles penetrating the outer membrane of B. malayi and deliver the drug directly the symbiotic bacteria Wolbachia.

As discussed above, the encapsulation and subsequent controlled release of ivermectin and doxycycline further enhanced our understanding of the ability of the polyanhydride nanoparticle carriers to localize to the worm cuticle surface and translocate into the worms, leading to a higher concentration of bioavailable drugs. Experiments detailed in chapter three continued the investigation of NP properties by examining the delivery of different cocktails of approved antiparasitic drugs against another Brugia filarial worm species, B. pahangi. B. malayi is a parasite of humans and does not infect rodents while B. pahangi is capable of causing disease in both host types. The animal models of lymphatic filariasis use B. pahangi infections in gerbils. In vitro, NP delivery of drug combinations diethylcarbamazine, albendazole, and doxycycline along with ivermectin and doxycycline provided significant benefits over soluble administrations.

The results showed that use of the NP as a delivery vehicle effectively killed adult worms, reduced microflorae shedding and acted as a microfilaricide. Attempts to perform in vivo studies were unsuccessful at demonstrating a significant difference due to the variability inherent in B. pahangi infections in gerbils and inability to conduct studies with larger numbers of animals. Additionally, future studies would need to be performed that focused on improving the pharmacokinetics of polyanhydride nanoparticles including; optimization of bioavailability, route of administration, and biodistribution to match the efficacy of the in vitro assays.

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The result from the Brugia chapters provides an understanding of the NPs behavior against intracellular bacteria. NPs are capable of delivering the antimicrobial doxycycline to the endosymbiotic bacteria Wolbachia and applied it to the intracellular bacteria

Mycobacterium. In chapter four we assessed co-encapsulation of the first line antitubercular rifampicin, isoniazid, pyrazinamide, and ethambutol with polyanhydrides.

The complexity of antimicrobial therapies directed against Mycobacterium tuberculosis result from the specificity of the intracellular nature of the bacteria. The innate abilities of the intracellular nature of Mycobacterium provide a challenging model to study the effectiveness of drug delivery. We sought to exploit the flexibility of NP properties to co-deliver small molecules intracellularly to treat Tuberculosis infections. A synergistic effect of NPs was demonstrated in chapters three and four to improve the effectiveness of approved anti-TB drugs was confirmed using in vitro culture and intracellular pathogenesis tissue culture models. The result showed numerous benefits using the nanoparticles in encapsulation and delivery of the four antituberculars effectively killing Mycobacterium in broth culture and subsequent studies treating macrophages.

We observed rapid and efficient internalization of NP by macrophages and intracellular distribution. The reduction of intracellular Mycobacterium in the presence of the nanoparticle suggests a possible co-localization event occurs between the bacteria. This theory supported by co-localization of NP with another intracellular bacterial pathogen,

Brucella. The most significant reduction in intracellular B. abortus and B. melitensis survival was observed in NP-treated groups that also exhibited the highest level of internalization by host macrophages. Microscopic observations revealed the bacteria colocalized with the NP [1-3] The macrophages treated with equivalent concentrations of the soluble

200 drug did not provide improved antimycobacterial activity. Future studies could include the examination of the bacterial/nanoparticle interaction by utilizing fluorescently labeled derivatives of selected carbohydrates that could functionalize to nanoparticles [3-5], providing insight into the interactions between Mycobacterium and the nanoparticles.

Studying of the bacterial interaction of mycobacteria with the carbohydrate functionalized nanoparticles could be accomplished through targeting of the intermembrane transporter system (ABC transporter). The intermembrane of Mycobacterium has been reported to possess five recognizable carbohydrate transporters [6] and shows the favorable use of host lipids as carbon sources for growth and persistence during infection [7]. Also,

Mycobacterium utilizes a substantial amount of carbohydrates as part of their unique cellular membrane components, which are carbohydrate-rich providing another alternative

[6, 7].

In chapter six we examined the ability of NP delivery of antitubercular drugs against select strains of multi-drug (MDR) and extensively drug-resistant (XDR) M. tuberculosis (TB). In the in vitro broth assay, polyanhydride nanoparticles improved the antimicrobial activity of soluble drugs against MDR-TB and XDR-TB. In infected macrophages, nanoparticles were able to enhance antimicrobial activity against intracellular

MDR-TB and XDR-TB. Similar results with Brucella and M. marinum suggests that the reduction in the bacterial population results from co-localization of the nanoparticle with both the MDR-TB and XDR-TB.

As previously mentioned the failures of most therapies occur during the transition from an in vitro model to an in vivo model. The research acquired from the in vitro study was applied to an acute TB infection in C3H mice, a model that mimics systemic disease

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[8, 9]. A component of this study quantified the number of persistent drug isolates recovered from infected tissues. The presence of a population of antibiotic persistent bacterial within the MDR-TB and XDR-TB treatment groups were found to be more significant after treatment with both the soluble and NPs comparatively, against the untreated. The early stages of infectious mycobacteria undergo constrained replication and are forced into a dormant state in the infected tissue by the host response. Bacteria were found to be rapidly replicating during early stages following infection compared to untreated. The stressor of antituberculars further exasperated the emergence of a persister or resistance population in the early infectious stages of the disease. The presence of an active persister population may suggest that the activity of the drug-loaded NPs provided a sufficient stress response to increase the cellular growth prematurely. Additional, studies are essential that focus on enhancing the pharmacokinetics including; bioavailability of the drugs, route of administration, and biodistribution of the polyanhydride nanoparticles. In general, encapsulation of antimicrobials in nanoparticles to treat mycobacterial infections could improve efficacy, lessen toxic side effects of prolonging treatment intervals all resulting in an improved patient compliance.

In conclusion, the studies carried throughout this dissertation has extended the volume of information regarding the use of amphiphilic polyanhydride nanoparticles as a nanotherapy against several infectious diseases. These remarkable results, suggests that the use of tailorable polyanhydride nanoparticles as a drug delivery vehicle. These studies reflect the need for new therapeutic approaches and facilitate bridging the gap between traditional infectious disease practices and the introduction of modern nanomedicine therapies.

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References

1. Goodman, J.T., et al., Nanoparticle Chemistry and Functionalization Differentially Regulates Dendritic Cell-Nanoparticle Interactions and Triggers Dendritic Cell Maturation.(Report). Particle & Particle Systems Characterization, 2014. 31(12): p. 1269.

2. Ulery, B.D., et al., Polymer chemistry influences monocytic uptake of polyanhydride nanospheres. Pharm Res, 2009. 26(3): p. 683-90.

3. Phanse, Y., et al., Functionalization promotes pathogen-mimicking characteristics of polyanhydride nanoparticle adjuvants. J Biomed Mater Res A, 2017. 105(10): p. 2762-2771.

4. Ross, K.A., et al., Nano-enabled delivery of diverse payloads across complex biological barriers. J Control Release, 2015. 219: p. 548-559.

5. Narasimhan, B., J.T. Goodman, and J.E. Vela Ramirez, Rational Design of Targeted Next-Generation Carriers for Drug and Vaccine Delivery. Annu Rev Biomed Eng, 2016. 18: p. 25-49.

6. Kalscheuer, R., et al., Trehalose-recycling ABC transporter LpqY-SugA-SugB- SugC is essential for virulence of <em>Mycobacterium tuberculosis</em>. Proceedings of the National Academy of Sciences, 2010. 107(50): p. 21761.

7. Niederweis, M., Nutrient acquisition by mycobacteria. Microbiology, 2008. 154(3): p. 679-692.

8. Kramnik, I. and G. Beamer, Mouse models of human TB pathology: roles in the analysis of necrosis and the development of host-directed therapies. Seminars in Immunopathology, 2016. 38: p. 221-237.

9. Hussein, D.A.E.-M., et al., Mycobacterium tuberculosis infection in systemic lupus erythematosus patients. The Egyptian Rheumatologist, 2017. 39(4): p. 227- 231.