Nanoencapsulation of Bilirubin and Its Effects on Isolated

NANOENCAPSULATION OF BILIRUBIN AND ITS EFFECTS ON ISOLATED

MURINE PANCREATIC ISLET CELLS

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Bronwyn Anne Fullagar BVSc (Hons)

Graduate Program in Comparative and Veterinary Medicine

The Ohio State University

2015

Thesis Committee:

Christopher A. Adin, Advisor

Chen Gilor

Alicia Bertone

Copyrighted by

Bronwyn Anne Fullagar

2015

Abstract

Pancreatic islet transplantation would provide a cure for type 1 diabetes mellitus in dogs, but isolation stress and hypoxia cause loss of up to 70% of islets in the first 72h after transplantation. Bilirubin (BR) is a natural antioxidant and can improve survival of murine pancreatic allografts exposed to hypoxic stress. However, its poor bioavailability limits its therapeutic application. Nanoparticle (NP) drug delivery, using biopolymers

Pluronic F127 and chitosan, can improve solubility and bioavailability of hydrophobic drugs such as bilirubin. We hypothesized that delivery of bilirubin via Pluronic F127- chitosan nanoparticles (nBR) would improve uptake into murine islets compared to free bilirubin (fBR). We further hypothesized that nBR would improve the viability of islets following exposure to hypoxic stress, compared to fBR or control.

Pluronic F127-chitosan NPs were synthesized and bilirubin was nanoencapsulated at a feeding ratio of 1:20. Murine INS-R3 cells, an insulinoma cell line, were incubated in media containing 0-20µM nBR, fBR or empty NP (eNP). After staining with Hoescht and LysoTracker Red, bilirubin uptake was qualitatively studied using Zeiss Apotome (confocal-like) structured illumination microscopy. Further INS-R3 cells were then cultured in media with nBR, fBR or eNP at concentrations of 0-20µM.

Cells were exposed to 8h hypoxia (1% O2), followed by 12h recovery (standard conditions). MTT viability assays were performed and cell viability was expressed as

ii percentage absorbance of control (untreated) cells.

In accordance with the IACUC, pancreatic islets were isolated from female

C57BL/6 mice and were incubated with nBR, fBR or eNP at 0-20µM, then exposed to

24h of hypoxia (1% O2). Cells were stained with Propidium Iodide (PI) and Hoescht and imaged using epifluorescent microscopy. Images were analyzed using NIH Image J software to determine the percentage of PI positive cells in each islet. Analysis of cell viability data was via the linear mixed procedure using SPSSv21 software. Significant main effects were investigated using Sidak multiple comparison tests; p≤0.05 was considered statistically significant.

Qualitatively, INS-R3 cells showed increased uptake of nBR compared to fBR at all concentrations and active, selective uptake via endocytosis was apparent at 20µM. In

INS-R3 cells exposed to hypoxic stress, cells treated with 5µM nBR (113.2% +/- 3.9), had the best viability overall and there was dose-dependent cytotoxicity in all groups. In murine islets exposed to hypoxic stress, islets treated with nBR survived significantly better than other groups (p=0.047). Islets treated with 5µM nBR (18.5% +/- 14.1) survived better than untreated islets (33.5% +/- 17.5%), with reduction of central necrosis. Dose-dependent cytotoxicity at 20µM was seen in all groups. The mechanism of the improved protective effects of nBR is likely due to active, selective uptake of NP via endocytosis.

Unencapsulated BR (fBR) had insignificant protective effects on murine islets exposed to 24h hypoxia, which may indicate that its protective mechanisms were overwhelmed by the prolonged hypoxic stress in this study. Central necrosis is a

iii characteristic feature of pancreatic islets exposed to hypoxic stress, due to impaired diffusion of nutrients. These effects appear to be ameliorated by treatment with nBR, which may target the metabolically active β-cells residing at the center of murine islets.

Empty NP appear to be cytotoxic, which may indicate a pro-inflammatory effect, as chitosan has been shown to induce an IL-1β response.

Pluronic F127-chitosan nanoencapsulation of bilirubin is feasible and results in improved cellular uptake and dose-dependent protective effects on murine islets exposed to hypoxic stress. Further investigation into the protective mechanisms of nBR and toxicity of empty NP are warranted.

This thesis is dedicated to my family, Peter, Nance and Andrea, for their constant love and support, and to my advisor, Dr. Adin, for his endless enthusiasm, encouragement and

exceptional mentorship.

Acknowledgments

This study was supported by a grant from the Paladin Research Fund. The author would like to acknowledge collaborators Dr. Wei Rao and Dr. Xiaoming (Sean) He at

The Ohio State University Department of Biomedical Engineering for their expertise in nanoparticle synthesis and experimental methods, Feng Xu for her technical expertise and patience in teaching me laboratory techniques and Dr. John Bongaura for his assistance with the statistical analyses.

Vita

2003-2007 ...... Bachelor of Veterinary Science, University of

Queensland, Brisbane, Australia

2011-2012 ...... Small Animal Rotating Internship, Calgary

Animal Referral and Emergency Centre, Alberta,

Canada

2012 to present ...... Graduate Teaching Associate and Small Animal

Surgery Resident, Department of Veterinary

Clinical Sciences, The Ohio State University

Fields of Study

Major Field: Comparative and Veterinary Medicine

vii

Table of Contents

Abstract…………………………………………………………………………………… ii

Acknowledgments……………………………………………………………………….. vi

Vita……………………………………………………………………………………… vii

Fields of Study…………………………………………………………………………... vii

List of Tables…………………………………………………………………………….xii

List of Figures…………………………………………………………………………...xiii

Chapter 1: Nanotechnology in veterinary surgery - applications and future directions………………………………………………………………………………….1

Introduction………………………………………………………………...... 1

Nanomaterials………………………………………………………………………….. 2

Organic (polymeric) nanoparticles………………………………………………….. 2

Inorganic nanoparticles……………………………………………………………… 3

Nanoparticle drug delivery…………………………………………………………….. 4

Drug nanocrystals…………………………………………………………………… 5

Water-soluble polymers……………………………………………………………... 5

Liposomes…………………………………………………………………………… 6

Polymeric nanoparticles……………………………………………………………... 7

Nanoparticle characteristics……………………………………………………………7

viii

Preparation of polymeric nanoparticles……………………………………………... 7

Particle size………………………………………………………………………….. 8

Surface properties…………………………………………………………………… 9

Drug loading……………………………………………………………………….. 10

Drug release………………………………………………………………………... 10

Triggered drug release……………………………………………………………... 11

Organ/cellular targeting……………………………………………………………. 12

Intracellular targeting………………………………………………………………. 13

Current and future applications in veterinary surgery……………………………….. 13

Surgical oncology………………………………………………………………….. 13

Thyroidectomy and parathyroidectomy…………………………………………… 18

Wound healing and burns………………………………………………………….. 19

Tendon and ligament injuries……………………………………………………… 21

Bone diseases and bone regeneration……………………………………………… 22

Hemostasis and trauma…………………………………………………………….. 23

Transplant………………………………………………………………………….. 24

Other applications in veterinary medicine………………………………………… 26

Nanoparticle toxicity…………………………………………………………………... 27

Conclusion……………………………………………………………………………. 29

Chapter 2: Nano-encapsulated bilirubin protects murine pancreatic islet cells exposed to hypoxic stress in vitro……………………………………………………… 31

Introduction…………..……………………………………………………………………… 31

Materials and methods………………………………………………………………………...34

Materials…………………………………………………………………………… 34

Synthesis of Pluronic F127-chitosan nanoparticles………………………………... 35

Encapsulation of bilirubin to obtain nanoparticle-encapsulated bilirubin (nBR)….. 37

Spectrophotometric analysis of bilirubin content within nanoparticles…………….38

In-vitro release studies……………………………………………………………... 38

Cellular uptake and intracellular distribution of nBR in INS-R3 cells…………….. 39

Effect of nBR on viability of INS-R3 cells exposed to hypoxic stress……………..41

Effects of nBR on viability of murine islets exposed to hypoxic stress…………… 41

Statistical analyses…………………………………………………………………. 42

Results………………………………………………………………………………… 43

Physiochemical characterization of nanoparticles…………………………………. 43

Bilirubin release characteristics……………………………………………………. 44

Cellular uptake of bilirubin in INS-R3 cells……………………………………….. 45

Effects of nBR on INS-R3 cells under hypoxic conditions………………………... 46

Effects of nBR on murine islets under hypoxic conditions………………………... 48

Discussion…………………………………………………………………………….. 51

Release characteristics of nBR and fBR…………………………………………… 51

Improved uptake of nBR by murine islet cells…………………………………….. 53

Effect of nBR on INS-R3 cells exposed to hypoxic stress………………………… 54

Protective effects of nBR on murine islets following hypoxic stress……………… 55

Toxicity of eNP to murine islets and INS-R3 cells…………………………………56

Negligible protective effects and dose-dependent toxicity of fBR………………… 57

Limitations and future directions…………………………………………………... 58

Conclusion……………………………………………………………………………. 58

References……………………………………………………………………………….. 60

List of Tables

Table 1: Encapsulation efficiency (EE) and loading content (LC) of bilirubin together with diameter of the resultant NP-encapsulated bilirubin (nBR), determined by dynamic light scattering (DLS): all data are presented as mean +/- standard deviation...... 44

xii

List of Figures

Figure 1: The protective effects of bilirubin during pancreatic islet transplant………….33

Figure 2: Procedure for synthesis of Pluronic F127-chitosan NP and for encapsulating a hydrophobic molecule, curcumin.16 ……………………………………………………..36

Figure 3: A) Scanning electron micrograph of Pluronic F-127 chitosan NP at room temperature; B) Dry nBR at room temperature (feeding ratio 1:20)…………………….37

Figure 4: Procedure for testing the release characteristics of nBR and fBR in solutions with or without protein…………………………………………………………………...39

Figure 5: Release of bilirubin (expressed as % of original bilirubin concentration) through a 20kDa dialysis membrane, suspended in dialysate with or without protein. Group 1: nBR in PBS+10% albumin; Group 2: fBR in PBS+10% albumin; Group 3: nBR in PBS; Group 4: fBR in PBS…………………………………………………………………….45

Figure 6: Uptake of nBR (bottom row of each image), fBR (middle row) and eNP (top row), at concentrations of 5µM (A), 10µM (B) and 20µM (C), by INS-R3 cells in culture……………………………………………………………………………………46

Figure 7: Mean viability (expressed as % of viability of untreated cells) of INS-R3 cells treated with 0-20µM nBR, fBR or eNP and exposed to 8h hypoxia…………………….47

Figure 8: Mean cell death (expressed as % PI positive cells) of murine islets treated with 0-20µM nBR, fBR or eNP and exposed to 24h hypoxia………………………………...49

Figure 9: Islet survival (expressed as % of initial number of islets) after treatment with 0- 20µM nBR, fBR or eNP and exposure to 24h hypoxia...... 50

Figure 10: Hoescht (nuclear, blue) and PI (cell death, red) staining of murine islets treated with 0-20µM nBR, fBR or eNP and exposed to 24h hypoxia. Images were taken using epifluorescent microscopy at 4x magnification...... 50

xiii

CHAPTER 1: NANOTECHNOLOGY IN VETERINARY SURGERY:

APPLICATIONS AND FUTURE DIRECTIONS

Introduction

Nanotechnology, defined as the control, manipulation, study and manufacture of structures and devices less than 1000nm in size1, has revolutionized human medicine over the past 30 years. Nanoparticles, which generally have a diameter of less than

200nm1,2, have far-reaching medical and surgical applications including tumor diagnostics, vaccines, chemotherapy, osteoarthritis, wound healing, transplant and hemostasis. Their main advantage lies in their ability to provide targeted delivery of drugs to specific cells or tissues, allowing for improved bioavailability (especially via the oral route), sustained effects in target tissues, delivery of insoluble drugs intravenously and protection of drugs from enzymatic breakdown.1,3,4 To date, most published literature focuses on applications for nanoparticles in human medicine. However, there is significant potential for the application of similar technologies to veterinary medicine and surgery.

Nanomaterials

When materials are manipulated at nanoscale, their chemical and biological properties differ fundamentally from those of the corresponding bulk materials.5 Their unique features, such as their increased surface to mass ratio, quantum properties and ability to adsorb and carry other compounds make them attractive for medical applications.2,6 The action, bioavailability and toxicity of nanoparticles is determined by factors such as their geometrical shape, surface coating and electrical charge. Awareness of the unique physiochemical characteristics is vital to maximize their therapeutic benefit and minimize toxicity to host tissues.

Nanomaterials can be broadly divided into two categories: organic or inorganic.

Organic nanomaterials can be further classified as biological (natural), or synthetic

(biocompatible/biodegradable) polymers.6-8 Natural polymers, including proteins or polysaccharides (e.g. gelatin, albumin), are used less frequently than synthetic polymers, due to variations in purity and rapid denaturation after administration.

Organic (polymeric) nanoparticles

The most commonly used synthetic polymers for nanoparticle synthesis are poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their co-polymer, poly(lactic-co- glycolic acid) (PLGA). These polymers are biocompatible and biodegradable, and have been tested extensively for toxicity and safety in animal models. They are currently FDA 2 approved for a number of human applications, including resorbable sutures and bone implants.3,9,10

Chitosan is a naturally-occurring, biodegradable polysaccharide, produced from chitin, which is derived from the exoskeletons of crustaceans. Alginate is a natuarally- occurring biopolymer derived from brown algae. Both are biocompatible, biodegradable and non-toxic. They are particularly suited to the oral delivery of protein pharmaceuticals, due to their resistance to low pH and mucoadhesive properties.10,11

Delivery of protein drugs via the oral route has traditionally been limited by protein degradation and denaturation by the acidic pH of the stomach. Nanoparticle chitosan and alginate can protect protein drugs from enzymatic degradation in the gastrointestinal tract

(GIT)12 and increase penetration of drug across mucosal surfaces, by bonding with mucosa via free carboxyl groups.11,13

Inorganic nanoparticles

In addition to drug delivery, inorganic nanoparticles, including gold, silver, platinum and iron oxide, are commonly used for imaging in tumor diagnostics. Iron oxide

NP are magnetic and their distribution and release characteristics can be influenced by application of a high-gradient magnetic field.

Quantum dots (QD) are semi-conductor nanoparticles that fluoresce when stimulated by light at wavelengths between 450 and 850nm.14 They are used to improve the sensitivity of molecular imaging and can be conjugated to various biomarkers, such as antibodies or peptides, for highly accurate disease detection. 5,6,15Inorganic NP, for 3 example, gold, magnetic or carbon, can also be heated using electromagnetic waves to treat cancer cells with targeted hyperthermia or radiation therapy.16-18

Nanoparticle drug delivery

Due to their sub-cellular and sub-micron size, nanoparticles have improved deep tissue penetration, cross epithelial fenestrations more readily and have more efficient cellular uptake, making them more efficient than traditional methods of drug delivery.3

Up to 95% of potential therapeutic agents in human and veterinary medicine have poor bioavailability; nanoscale delivery systems can be engineered to improve therapeutic index, reduce toxicity, confer controlled release and deliver these drugs to targeted sites by intravenous, oral or minimally-invasive routes.3,15 In addition, drug release characteristics can now be tailored to a specific disease process and method of administration, with nanoparticles responding to biological stimuli such as temperature, light, magnetic field, pH and oxidative stress.10

In food-producing animals, increased efficiency of drug dosing allows reduction of drug residues in carcasses, leading to decreased environmental and public health concerns. Since many human-approved drugs are prohibitively expensive for veterinary patients, especially large animals, improved therapeutic indices may allow their use in veterinary medicine. Nanoparticle delivery systems can also offer enhanced performance

4 of an existing drug at about 20% of the cost and less than half the development time as developing a new drug.19

The most popular nanodrug delivery systems currently in use in human and veterinary therapeutics are drug nanocrystals, water-soluble polymers such as dendrimers and micelles, liposomes and polymeric nanoparticles.4,6,9-11

Drug nanocrystals

Nanocrystals are composed purely of drug, with a particle size of 10-1000nm; there is no carrier material as in other forms of NP. When dispersed in media, usually water, aqueous solution or non-aqueous solution (e.g. liquid polyethylene glycol [PEG]), they form a ‘nanosuspension’. Approximately 60% of all new drugs released in human therapeutics are insoluble in water, which correlates to poor bioavailability and poor absorption from the gastrointestinal tract into the blood stream.20 Nanocrystals create more uniform and predictable bioavailability of drugs with poor aqueous solubility, by increasing dissolution velocity through surface area enlargement and increasing saturation solubility.4,20

Water-soluble polymers

Polymer-drug conjugates are nanostructures where a drug or biologically active protein is covalently bound to a water-soluble polymer carrier. Examples of common carrier molecules are polyethylene glycol (PEG), polyglutamate and albumin. These carriers are non-toxic to cells and non-immunogenic, allowing them to be degraded and

5 eliminated easily from a patient. Antibodies specific to a certain disease, or stabilizing groups, can be added to the polymer backbone to alter the bioavailability of the conjugate.4

Dendrimers are monodisperse macromolecules with a branching, three- dimensional architecture. Their unique structure confers properties different from linear drug counterparts. Dendrimers can act as vectors in gene therapy, due to their ability to transfer a large amount of genetic material to the cell nucleus.4

Micelles are characterized by a hydrophobic core, stabilized by a hydrophilic shell, and are formed by the self-assembly of amphiphiles in water.4,15 They allow delivery of hydrophobic drugs and are safe to be administered parenterally, making them ideally suited to oral and mucosal drug delivery.

Liposomes

Liposomes, or phospholipid vesicles, possess a central aqueous cavity surrounded by a membrane of concentric lipid bilayers.4 They are versatile in their ability to carry both hydrophilic and hydrophobic drugs and can be administered topically, IV or IM, but not orally.15 They have been used for administration of chemotherapeutics, vaccines, imaging markers and nucleic acid-based therapies, but are limited by their low encapsulation efficiency, poor storage stability and rapid leakage of water-soluble drugs into the bloodstream.9

Polymeric nanoparticles

Polymeric nanoparticles are defined as solid, biodegradable particles predominantly between 20 and 100 nm in diameter that are prepared from natural or synthetic polymers, most commonly PLA and PLGA.7,9 Drugs can be incorporated into nanoparticles by a number of mechanisms, including entrapment in a polymer matrix

(nanospheres), encapsulation within the NP core (nanocapsules), surrounded by a shell- like polymer membrane, chemically conjugated to a polymer or bound to their surface by adsorption.9 Polymeric NP are more stable than micelles or liposomes and allow for controlled drug release. Their applications include biological markers, imaging, drug delivery and simultaneous detection, diagnosis and treatment of various diseases

(theranostics). As drug delivery devices, they can be targeted to specific cells of interest and can carry lipophilic drugs, proteins, peptides and genes for oral or mucosal administration.4

Nanoparticle characteristics

Preparation of polymeric nanoparticles

A comprehensive review of the methods by which NP can be produced is beyond the scope of this review. Briefly, two main approaches can be used. One involves the use of preformed polymers, whereas the other utilizes monomers that are polymerized in situ.

Various preparation methods exist, depending on the physical and chemical properties of

7 the polymers and monomers used. Usually, two main steps are involved. Firstly, a dispersed or emulsified system is prepared, containing drug and polymer components.

Next, polymers are precipitated or dispersed from the emulsion to form NP. In some cases, an external coating (e.g. PEG) is added to improve biocompatibility. In order to prepare NP for therapeutic administration, they may require sterilization via filtration and are dried into a powder, via lyophilization or spray-drying.1

Particle size

Absolute particle size and size uniformity are the most important characteristics in determining in vivo distribution, cellular uptake, drug loading, toxicity and targeting ability of nanoparticles. In general, smaller nanoparticles have greater surface area, which leads to greater cellular uptake and faster release of drug.1 The reported optimal size for both cellular uptake (by endocytosis) and systemic drug delivery is 10-30nm.16 NP of

100nm or less would be ideal for rapid dissolution and arterial uptake, whereas larger NP

(~800nm) might be used for prolonged circulation time, or if the mononuclear phagocytic system was the target.9 This has clinical implications in that smaller NP are able to cross mucosal layers (e.g. olfactory or alveolar epithelium) and the blood-brain-barrier, enabling them to deliver chemotherapeutics to previously isolated regions of the body.

However, these characteristics also play a role in the toxicity of NP, since they can be distributed systemically and retained within the circulation, leading to long-term inflammation and cellular oxidative damage.2

Surface properties

Nanoparticles are recognized by the host immune system and are subject to phagocytosis by mononuclear cells, resulting in degradation before they reach their target site. The hydrophobicity of their surface determines the level of opsonin binding and non- coated (hydrophobic) NP are rapidly opsonized and cleared by the reticuloendothelial system.1,2 Polyethylene glycol (PEG) is a biodegradable co-polymer with hydrophilic characteristics. Coating NP with PEG creates a repellent layer that impedes their interaction with blood opsonins and suppresses macrophage recognition, prolonging their time in circulation.1,2,4

In addition to surface coating, zeta potential is an important determinant of NP stability and cellular uptake. The zeta potential is a measure of the surface charge of the particle, where a larger absolute value of the zeta potential indicates a larger charge. NP with larger surface charges will have stronger repulsive interactions and therefore will be more stable, with a more uniform size distribution. A zeta potential of +/- 30mV is required to create a physically stable nanosuspension and prevent NP aggregation. PEG surface modification lowers negative zeta potential, increasing particle stability.1,4,9

Charge can also aid in uptake of NP by the patient, especially via the GIT. Positively charged chitosan NPs loaded with antimicrobials were able to interact with the negatively charged sites on gastric mucosa, which opened the tight cellular junctions and allowed for treatment of Helicobacter pylori infection.12

Drug loading

In order to successfully deliver drug to its target tissue, NPs must first be loaded with therapeutic agent. A high encapsulation efficiency, or drug-loading capacity, is preferable, since this minimizes the absolute number of NP and hence the dose of NP matrix compounds administered to the patient.1 This can be achieved by two methods: incorporation, where drug is incorporated (or encapsulated) within the NP at the time of

NP formation, or absorption/adsorption, where formed NP are incubated in a concentrated drug solution. NP drug loading and encapsulation efficiency is greatest when drugs or proteins are loaded close to their isoelectric point. Encapsulation efficiency also increases with increasing NP diameter.1,9,15

Drug release

One of the most important features of polymeric NP is their potential for sustained drug release. The release rate of entrapped drug is affected by both drug dissociation from the NP and degradation of the NP polymer matrix. Thus, drug solubility within the matrix, desorption of the adsorbed (surface-bound) drug, drug diffusion through the NP matrix and NP matrix erosion all play a role in determining release characteristics.1,3,9 Most NP systems will release drug with an initial ‘burst’ over

24-48 hours, thought to be caused by release of adsorbed drug from the outside of the particles. This is then followed by a period of more sustained release.21 NP with lipophilic coatings can reduce the initial ‘burst’ and result in more sustained release,

10 whereas smaller particles with higher encapsulation efficiency will speed drug unloading.1,6,9

Triggered drug release

In order to regulate the release of drug for targeted delivery to the tissue of interest, NP can be engineered to respond to stimuli from the patient or environment.10

For example, differences in pH can ‘signal’ a different location within the gastrointestinal tract (pH 1–8.2), entrance into a cell via endocytosis (endosomal pH is 5–6.5) or the presence of ischemia or tumor cells in the nearby tissue (pH 6.5–7.2).10 Chitosan-alginate

NP shrink in size in response to the low pH of the stomach and release less drug into the gastric lumen, resulting in improved cellular uptake of nano-encapsulated drug by enterocytes and hence increased drug absorption into the bloodstream6,11

Temperature can also affect NP size, charge and release characteristics. Pluronic

F127-Chitosan NP have significantly larger diameter in colder temperatures (4oC) than at body temperature (37oC) and therefore showed much higher drug release at lower temperatures.22 Similarly structured NP, containing the anti-cancer drug curcumin, were also found to have significantly improved uptake by human prostatic adenocarcinoma cells at 43oC compared to at 37oC or 22oC.16 These properties allow for temperature- controlled drug release, through the application of thermo- or cryotherapy to the patient.

Hyperthermia itself can also be used to kill tumor cells. Treating the tumor first with iron oxide NP or gold nanorods, which will selectively accumulate within tumor cells, then applying an external high frequency magnetic field or infrared laser, can result in

11 minimally invasive, selective tumor ablation, without damage to normal tissues.14,17,23

Organ/cellular targeting

Targeted drug delivery to specific tissues or cells is an important benefit of NP delivery systems, for two reasons. Firstly, NP can deliver a concentrated dose of drug to a specific site, such as a tumor, and secondly, NP would reduce the dosing of drug to healthy tissues, thus minimizing toxicity.4 Targeting at the tissue or cellular level can occur via two mechanisms: passive targeting and active targeting.

Passive targeting is achieved via the innate properties of the NP conferred by their small size. The typical mechanism is via the ‘enhanced permeability and retention’ (EPR) effect. This refers to the propensity of NP to extravasate selectively at sites of increased vascular permeability, such as a focus of inflammation, infection or neoplasia, but otherwise to remain in the circulation (sometimes referred to as ‘stealth particles’).1,4,15

Tumors produce vascular endothelial growth factor (VEGF), which promotes disorganized angiogenesis, leading to the production of ‘leaky’ blood vessels. In addition, poor lymphatic drainage from the tumor site results in accumulation of drug-laden NP.23

These effects facilitate selective NP drug distribution to neoplastic tissue.

Active targeting can further increase the interaction between NP and their target tissues. This involves molecular recognition processes like ligand-receptor or antigen- antibody binding. NP can be targeted to inflammatory markers, adhesion molecules and abnormal cell surface receptors. For example, folate receptors are overexpressed on malignant cells, so NP bearing folate ligands may be used to target cancer.4 Ligand- 12 mediated binding can also facilitate endocytosis, for drugs that will not readily diffuse across cell membranes and require active uptake.

Intracellular targeting

Once a NP drug has reached its target tissue, it can enter cells via phagocytosis, fluid-phase pinocytosis or endocytosis. Cellular uptake is influenced by drug concentration and NP size (10-30nm is optimal for endocytosis).2,3 The surface charge of the NP also determines their fate within the cell. Effective intracellular targeting to specific organelles is important to maximize drug delivery, and can be demonstrated using organelle-specific stains.22

Current and future applications in veterinary surgery

Surgical oncology

Nanoparticle drug delivery has been used extensively for the targeted delivery of chemotherapeutics in order to achieve high concentrations within tumor cells, while sparing normal tissues. Although a comprehensive review of NP use in the treatment of cancer is beyond the scope of this article, NP have important applications in surgical oncology, allowing less invasive cancer staging and pre-surgical imaging of solid tumors.

Advanced imaging – MRI, nuclear scintigraphy, CT and PET – can be enhanced by the use of either NP with innate imaging properties (gold NP, quantum dots) or NP loaded

13 with contrast agents (liposomes), which are taken up selectively by tumor cells.14 This allows not only accurate imaging of the primary tumor and metastases, but also the accumulation and retention of NP within the tumor can be assessed non-invasively.24

Nanoparticle-enhanced imaging has the additional benefit of the potential to treat the tumor simultaneously, through delivery of chemotherapeutics, hyperthermia or radiation – a process known as theranostics. Iron oxide magnetic nanoparticles (MNP) exhibit superparamagnetic behavior, where thermal energy can spontaneously change the magnetization within each MNP by temperature influence. This allows for the delivery of hyperthermia selectively to tumor cells.16,17,24,25 Magnetic hyperthermic therapy (MHT) has been reported for human glioblastoma, using superparamagnetic iron oxide NP

(SPIONs). This involves injection of iron oxide nanoparticles inside the tumor, followed by placement of the patient in an alternating magnetic field, which results in an increased intratumoral temperature, thermal ablation of tumor cells, and subsequent tumor shrinkage.17

In humans, nanoparticle-enhanced imaging has been reported for the diagnosis and treatment of malignancies, including prostate cancer26, colorectal cancer27, breast cancer23, esophageal cancer6 and glioblastoma17.

Pre-surgical imaging

In human as well as veterinary oncological patients with solid tumors, there is a need for accurate, three-dimensional imaging of the extent of the primary tumor.

Advanced imaging allows pre-surgical planning of surgical margins, as well as determining whether a minimally invasive approach for tumor excision is feasible.

For example, magnetic nanoparticles (MNP) have been used for MRI imaging of human breast cancer.23 In addition to passive targeting via EPR, active targeting of MNP can be achieved through conjugation with monoclonal antibodies to cell surface HER2 receptors, which are present in about 17% of human breast cancers. HER2 antibodies on the surface of SPIONs can also be conjugated to fluorescent probes, to allow tumor imaging via both fluorescence imaging and MRI.23 The mechanism by which SPIONs enhance MRI imaging is via enhancement of proton spin–spin (T2/T*2) relaxation, causing reduction in signal intensity – negative contrast – on T2 weighted sequences.24

Imaging using SPIONs is also used for brain tumors such as glioblastoma, where obtaining accurate identification of the extent of cancer is paramount before considering surgery.17,24

NP-enhanced MRI would be particularly useful for pre-surgical planning in veterinary patients with locally invasive solid tumors such as soft tissue sarcoma, canine oral fibrosarcoma and feline injection site sarcoma. Application of this technique may provide a means to plan surgical margins more accurately. Further research into specific surface receptors of such tumors, as well as wider availability of MRI in veterinary institutions, is required before such practices can become mainstream in veterinary specialty practice.

Tumor staging

Staging provides important prognostic information and also helps to guide cancer therapy, both medical and surgical. In veterinary cancer patients, a systematic approach is achieved by using the ‘‘tumor node metastasis’’ (TNM) system, to evaluate the tumor, 15 the regional lymph node, and any possible distant metastases.28 Lymph node staging involves lymph node palpation, imaging, fine needle aspiration and/or lymphadenectomy.

Lymph node size alone has been found to be a relatively poor predictor of metastatic spread. In a study of 47 dogs with oral malignant melanoma, sensitivity and specificity of lymph node size as a predictor of metastasis were 70 and 51%, respectively, and the positive and negative predictive values were 62 and 60%, respectively.29 However, nodal aspiration or biopsy is sometimes challenging in nodes which are not peripherally accessible, and some tumors metastasize to unpredictable node locations.23 Thus, there is a need for a non-invasive method for determining nodal metastasis that is more accurate than size alone.

To this end, lymphotrophic nanoparticle-enhanced MRI (LN-MRI) has recently been reported for staging of human prostate cancer.26 Lymph node size on MRI has a reported sensitivity and specificity of 45.4% and 78.7% for this tumor. Chernyak et al described the intravenous administration of biodegradable SPIONs. The NPs were selectively phagocytosed by macrophages of the reticuloendothelial system (RES) of normal lymph nodes, while they were not taken up by the abnormal RES of metastatic nodes. NP uptake resulted in a decrease in signal intensity of normal nodes, making the metastatic nodes comparatively brighter on T2 and T2* weighted images. The resultant sensitivity and specificity were 100% and 95.7%, respectively.26

This technique could be applied to conditions such as canine and feline oral tumors and canine apocrine gland adenocarcinoma of the anal sac, where lymph node

16 staging has important prognostic and therapeutic importance, but where lymph nodes are not easily accessible for fine needle aspiration.30

Another method of lymph node staging in human cancer patients is identification and biopsy of the sentinel lymph node (SLN), the first tumor-draining node, which helps to determine a plan for local and regional tumor control.31 However, it can be challenging at surgery to visually identify sentinel nodes, especially if they are of normal size.

Traditionally, lymphoscintigraphy using radiocolloids such as 99m Tc-NanoColl has been used to plan the surgical biopsy procedure, then additional injections of fluorescent or colored dyes, along with acoustic gamma probe detection are commonly performed to provide real-time surgical guidance.23,31 A newer approach is the use of nanosized bimodal imaging agents, which combine radioactive and fluorescent properties within a single particle. The NP are injected into the primary tumor and are taken up selectively by SLN. This has the advantage of permitting three-dimensional surgical planning, as well as accurate intra-operative two-dimensional navigation based on fluorescence, from a single NP injection.31 An alternative method for SLN identification utilizes carbon NP injections into the site of the primary tumor. When carbon NP were injected into human colorectal cancers, the NP were selectively taken up by cells within the lymph nodes and invoked a color change that could be visualized laparoscopically.27 Following laparoscopic radical resection and lymphadenectomy, histopathology revealed a sensitivity of 91.7% and specificity of 100% for metastatic LN detection using this technique.27

In veterinary medicine, lymphoscintigraphy using NP bimodal imaging, or local tumor injection with dyed NP, would allow for excisional biopsy of sentinel nodes, with reduced morbidity associated with extensive dissection. A similar technique may be feasible for intra-operative imaging of diseases such as canine and feline chylothorax, in which identification of the thoracic duct via both fluoroscopy and direct visualization is advantageous.

Thyroidectomy and parathyroidectomy

Hypocalcemia due to removal of parathyroid glands is a common complication of thyroid surgery in both humans and small animals, particularly when bilateral thyroidectomy is performed. Incidence of hypocalcemia following bilateral thyroidectomy is 1.6%-50%32 in humans and <6% in cats33. In dogs, 25-47% of thyroid carcinomas occur bilaterally and 11 of 15 dogs in one study developed hypocalcemia following bilateral thyroidectomy.34 Preservation of normal parathyroid tissue during surgery while resecting hyperfunctional or neoplastic thyroid gland is challenging in all species, even for experienced surgeons, and numerous intra-operative imaging techniques have been reported. These include intra-operative frozen histopathologic sections of excised tissue, parathyroid scintigraphy, PTH chemiluminescent assay and intravenous methylene blue injection.32,33 However, Technetium-99m-sestamibi scintigraphy has poor sensitivity and specificity in dogs and methylene blue injection can cause Heinz body anemia and acute renal failure.33 Thus, there is a need for a novel method of intra- operative parathyroid gland identification.

Recently, Huang et al32 investigated an intra-operative, local injection of carbon

NP suspension for differentiation between thyroid and parathyroid glands in a prospective, randomized, controlled trial of human patients with thyroid gland neoplasia.

Carbon NP suspension (particle diameter 150nm) was injected into the cranial and caudal poles of the affected thyroid gland. This resulted in black staining of tumor and metastatic lymphatic tissue, while leaving the parathyroid glands unstained. The size of the NP prevented them from entering capillaries and the systemic circulation, since the capillary diameter is 30-50nm. Postoperatively, the incidence of clinical hypocalcemia and muscle cramps was significantly lower in the subjects who received carbon NP injection, compared to controls.32 There is the potential for this technique to be applied to dogs with thyroid carcinoma and cats with hyperthyroidism, for the preservation of parathyroid tissue.

Wound healing and burns

The application of topical antimicrobials can be used to reduce the bacterial burden on a wound surface, especially in situations where systemic vasoconstriction and local vascular compromise prevent systemic antimicrobials from reaching therapeutic levels. Silver is a commonly used topical antimicrobial, due to its effectiveness against resistant organisms.35 However, challenges exist in immobilizing silver at the wound surface, to maximize its local effect while avoiding systemic toxicity.36 Nanocrystalline silver dressings (e.g. ActicoatTM [Smith and Nephew]) release clusters of nanoscale, highly reactive silver particles. Nanocrystalline dressings provide improved silver

19 solubility, sustained delivery of silver ions, and have been shown to achieve a more rapid rate of bacterial kill in vitro. Animal studies suggest a role for nanocrystalline silver in altering wound inflammatory events, by modulating collagen deposition, and facilitation of the early phase of wound healing.37,38 Nanocrystalline silver has been widely used in human chronic wounds and burns and its use has been reported in combination with subatmospheric pressure wound therapy in a cat, for the successful treatment of a large, infected wound secondary to injection site sarcoma excision.35

Nitric oxide (NO) is a lipophilic, reactive effector molecule important in modulating all phases of wound healing – inflammation, proliferation and remodeling.

Chronic wounds are more common in both human and veterinary immunocompromised patients, such as those with advanced age, endocrinopathies or previous chemotherapy. In these patients, NO production can be decreased, due to reduced numbers of immune cells.

Blecher et al reported the use of NO-NP, made from chitosan with PEG coating, for topical treatment of wounds in non-obese, diabetic, severe combined immunodeficiency

(SCID) mice. NO-NP treatment accelerated wound closure, compared to controls treated with a topical synthetic NO treatment (DETA NONOate). Histologically, wounds treated with NO-NP had less inflammation, more collagen deposition and improved angiogenesis compared to other groups.39

There is great potential for the application of topical nanoscale treatments to veterinary patients with chronic wounds or burn injuries. Open wound management in animals can be costly, time-consuming and can lengthen hospital stays. Dressing changes frequently need to be performed under sedation or anesthesia. Therefore, the

20 development of technologies that more efficiently delivery topical drugs and speed wound healing, especially in immunocompromised patients, would greatly improve patient care and reduce owner expense.

Tendon and ligament injuries

Tendon and ligament injuries, including sprains, strains and rupture, represent a significant problem for both humans and animals, especially in canine and equine athletes. Lack of robust blood supply and low metabolism in mature tissues render ligament and tendon injuries prone to incomplete and prolonged healing, leaving patients with reduced mobility and prone to reinjury.38,40 As such, considerable research has been undertaken to develop a less invasive treatment approach, with improved functional outcome while reducing healing time and complications.

Nanoparticles have been shown to enhance the mechanical properties of natural polymer matrices such as chitosan and regenerated cellulose40 and nanofibrous materials that mimic extracellular matrix have been developed as tissue-engineered scaffolds for the skin, bone, vasculature, heart, cornea, nervous system, and other tissues.41 Empson et al40 investigated the feasibility and effect of using high stiffness nanocarbon and nanocellulose to reinforce damaged patella tendon in a rat model. NP-treated tendons had higher elastic moduli and yield strength than untreated tendons and fibroblast activity was higher in the treated group, without signs of toxicity.40 Silver NP, with their novel physiochemical properties, have also been investigated in the repair of experimentally transected rat Achilles tendons.38 Six weeks after treatment, AgNP-treated tendons 21 showed a significant improvement in tensile modulus compared to the untreated group and histopathology suggested that silver NP promoted cell alignment and proteoglycan synthesis.38

The above studies suggest that there is a role for nanoscale technology in the treatment of tendon and ligament injuries in both human and veterinary patients. Further research, in the form of in vivo studies of naturally-occurring injuries, as well as studies to investigate the long-term effects of NP on tendon strength and function are required to validate the techniques. Since canine and equine patients serve as a model of similar injuries in humans, there may be a role for veterinary clinical trials in this area.

Bone diseases and bone regeneration

Targeted drug delivery using nanotechnology has been widely proposed as a potential strategy for treating diseases of bone, such as osteoarthritis, osteosarcoma, metastatic bone tumors and osteomyelitis.42,43 There is a paucity of available effective treatments for these diseases and a balance between treatment efficacy and medication side effects must continually be sought. NPs have the potential to carry drug to its target destination at high concentrations, while preventing drug degradation and improving solubility. Improved efficacy of bisphosphonates and chemotherapeutics such as doxorubicin have been reported using NP drug delivery for the treatment of osteosarcoma and bone metastasis, both in vitro and in animal models.43 In addition, NP have been shown to increase the retention time of drugs such as IL-1Ra within joints, through ionic interactions with synovial fluid.43

In another study, lipid NP modified with chondroitin sulfate and carrying the antiosteoarthritic drug diacerein were delivered orally to rats with experimentally induced osteoarthritis.44 Drug uptake was significantly higher in the articular cartilage of NP- treated rats than controls. Since toxicity related to conventional treatments for osteoarthritis, such as NSAIDs, in dogs, cats and horses is of significant concern, nanotechnology may have the potential to significantly increase anti-arthritic drug concentration in joints while reducing the risk of systemic toxicity.

Hemostasis and trauma

Trauma is the leading cause of death of humans between 1 and 44 years old and most early trauma deaths are due to exsanguination/hemorrhagic shock (60-70%) or CNS injury.45-47 Trauma accounts for over 10% of veterinary hospital admissions and mortality rates of 10-14% are reported.46,48,49 Approximately 22-43% of dogs sustaining blunt trauma develop hemoabdomen46,48 and hemorrhagic shock is a common manifestation in canine trauma.46-48 Coagulation abnormalities commonly occur in both humans and dogs secondary to trauma due to acute traumatic coagulopathy (ATC), and are associated with a four-fold increase in mortality rate in humans.48 Thus, in both human and veterinary medicine, there is a need for hemostatic treatment that can be provided in the field or the emergency room, in order to prevent fatal hemorrhagic shock.45,50

Experimental animal trauma models have been evaluated to test novel therapies.

Recently, Shoffstall et al45 reported the use of hemostatic NP in a rat lethal liver resection injury model. The ‘synthetic platelets’, synthesized from NPs conjugated with a platelet

23 ligand, are composed of biodegradable PGLA and poly-L-lysine (PLL) with a PEG coating and were administered IV immediately following liver lobectomy. NP administration improved survival at 1 hour post injury from 40-47% in controls treated with inactive NP and saline, to 80%.45 Therapeutic effect was proposed to be due to preferential aggregation of NP at the site of hemorrhage and reduction in clotting time, while maintaining clot strength. A later study found that increasing the platelet ligand

(GRGDS) density by 100-fold resulted in further improvement in survival to 92%, compared to 45-47% in controls, at one eighth of the initial dose.50 Since higher concentrations of NP raise concerns about potential aggregation and pulmonary thromboembolism, improving dosing efficiency is of key importance.

Although laboratory animal models are frequently used for human trauma studies, they cannot reproduce the conditions of naturally-occurring trauma. Therefore it has been proposed that pet dogs with naturally occurring traumatic injury might represent a promising translational model for human trauma that could be used to assess novel therapies.47 Since the high cost of nanoscale hemostatic treatments is currently prohibitive for widespread veterinary use, clinical trials in client-owned dogs may represent the next step in trauma research, which could ultimately benefit both canine and human patients.

Transplant

Successful organ transplant in both humans and animals relies on effective suppression of the host’s immune response, in order to prevent allograft dysfunction and

24 rejection. A challenge in administration of immunosuppressive drugs is their low solubility and poor bioavailability.51 In addition, non-specific suppression of the entire immune system can be harmful to both the host and the transplanted tissue, resulting in complications such as increased susceptibility to infection and increased rates of neoplasia. Despite advances in immunosuppression protocols for human liver transplant recipients, five-year survival rates are still less than 80%.52

Nanoparticle-based delivery systems for hydrophobic immunosuppressive drugs have been recently reported. NP can improve drug solubility and bioavailability, especially via the oral route, provide targeted uptake by selected cells to obtain higher plasma concentrations and allow for sustained drug release.52 Xu et al investigated the delivery of tacrolimus encapsulated in a PEG-PLA biodegradable NP, using a rat liver transplant model. They reported sustained drug release and improved survival in rats treated with tacrolimus NP, compared to oral tacrolimus capsules.52 Similarly, Dane et al found that tacrolimus and rapamycin micelles prolonged allograft survival by twofold, when administered into local lymph nodes in a mouse tail skin allograft model.51 Nano- encapsulation of immunomodulatory drugs such as tacrolimus has the potential to reduce toxicity in veterinary patients, allowing more effective immune suppression in dogs and cats undergoing organ transplantation.

Donor tolerance can also be achieved by conditioning the recipient pre- and post- transplant with infusions of donor cells. More recently, administration of synthetic NP conjugated to donor antigens has been suggested.53 Bryant et al investigated PLGA NP as a cell-free carrier for donor antigens (PLG-dAg) for MHC-mismatched murine allogenic

25 pancreatic islet transplantation. Combining PLG-dAg with peritransplant rapamycin resulted in tolerance efficiency of ~60%, which was greater than the tolerance provided by PLG-dAg alone.53

Other applications in veterinary medicine

Outside of veterinary surgery, there are numerous other applications for NP in veterinary science. A recent review article contains an overview of nanoparticle systems evaluated in veterinary species for drug and vaccine delivery.15 NP delivery of vaccines has been reported for Rhodococcus equi54, rabies55, Enterotoxigenic E. coli56 and peste des petits ruminants57, among others. NP allow vaccines to be administered by novel routes (oral, nasal), which can improve their efficacy and allow more consistent dosing of extensively raised animals and wildlife.4 Targeted delivery of antibiotics has been applied to the treatment of biofilms on veterinary devices. Liposomes, polymeric NP and inorganic NP have been investigated and have the advantages of slow clearance rate and can deliver antibacterials at high concentrations directly to the biofilm interface and to phagocytes harboring intracellular pathogens.58 NP have been investigated extensively in the treatment of human cancer, and dogs and cats with spontaneous tumors represent an ideal model for many human neoplasms. Phase 1 clinical trials of NP-encapsulated chemotherapeutics for canine and feline cancer have been undertaken for hemangiosarcoma59, lymphoma60 and solid tumors61,62.

Nanoparticle toxicity

Nanoparticles offer unique physiochemical properties, which render them particularly useful to a wide variety of veterinary surgical situations. However, toxicity associated with their use will be a decisive factor in determining their widespread application. Not only must we consider potential toxicity to the animal patient, but also to humans who administer the NP, as well as residues in food-producing livestock.15 Four possible mechanisms of NP toxicity have been proposed: direct chemical toxicity of the

NP constituents, toxicity of degradation products, toxicity resulting from NP endocytosis and NP-related cell membrane lysis.2 Many NPs, especially inorganic or synthetic forms, contain elements such as gadolinium, cadmium, selenium, tellurium, arsenic and lead which are known to have acute or chronic toxicity in vertebrates.8,31 The increased surface area of NP and sustained release properties can result in more potent adverse effects than if the patient were exposed to the ‘bulk’ chemicals.2,8

Inhalation of particulate matter <10µm is known to lead to pulmonary inflammation and neoplasia. Toxicity of inhaled NP is inversely proportional to size, since smaller particles have a higher surface area and therefore increase the body’s exposure to pro-inflammatory chemicals. In addition, pulmonary inflammation may increase alveolar membrane permeability, which may allow NP to distribute beyond the lungs.2 Aggregation of NP after administration can result in emboli which may lodge in

27 the brain or lungs.9,31 NP have also been shown to cause cellular oxidative stress, by generation of surface radicals, and can affect mitochondrial function. They have been found to inhibit macrophage phagocytosis and can cause platelet aggregation.2

NP can gain access to the brain either via trans-synaptic transport following inhalation, through olfactory epithelium, or by crossing the blood brain barrier. Oxidative stress within the brain has been linked to neurodegenerative conditions such as

Parkinson’s and Alzheimer’s diseases in people. Evidence is still lacking as to whether

NP are implicated in the development of these diseases.2

NP absorption, metabolism, excretion and accumulation are dependent on NP size and constituent materials. NP with a diameter of <5nm quickly extravasate across the endothelium and have short blood circulation times.23 Although some small molecules can be excreted from the body through salivary glands and sweat, the majority of NPs clearance from the bloodstream is through either renal or hepatic routes.8 NP with a diameter of <6nm have a short blood circulation time and are filtered through urine, while those >8nm have longer half-lives and are phagocytosed by liver Kupffer cells, then undergo clearance via the biliary system. However, some NP may escape opsonisation by the reticuloendothelial system and can be retained within the systemic circulation, which may lead to toxicity.

Toxicity of polymeric nanoparticles has been investigated in several in vitro models.63 Nanocapsules composed of PLGA, PVA and Mygliol® 812 (Cremer Oleo,

Hamburg, Germany) did not present cellular toxicity. In an in vitro study of alginate- chitosan-pluronic composite NP, doses of up to 1mg/mL were associated with minimal

28 cytotoxicity to human cervical cancer cell lines (HeLa) in culture.21 On the other hand, murine islet capillary endothelial cells exhibited greater leukocyte adhesion at doses of

50µg/mL of empty PLGA-PEG NP, indicating pro-inflammatory activity of the NP themselves.64

In the process of developing novel NP drug delivery and diagnostic systems for veterinary patients we must carefully evaluate the potential toxicity of the NP themselves and distinguish them from the toxicities caused by the drug they contain. In vitro testing may not replicate the body’s response to NP in vivo, especially in patients with pre- existing disease. A thorough understanding of the unique biological responses to nanomaterials is needed to develop and apply safe drug delivery systems in future.

Conclusion

Although the use of nanotechnology is not yet mainstream in veterinary surgery, there is great scope for future research in to its application, especially in the fields of intra-operative imaging and regenerative medicine. Since dogs and horses serve as excellent models of many human diseases, experimental and clinical trials involving novel NP therapies could pave the way for future improvements in disease diagnosis and treatment for humans and animals alike. There is still uncertainty regarding the long-term safety of nanoscale drug administration and careful investigation of cellular, tissue and systemic effects of novel treatments with follow-up is crucial. Essential to the provision

29 of safe nanodrugs will be effective regulation of the nanotechnology industry for both human medical and veterinary fields.

CHAPTER 2: NANO-ENCAPSULATED BILIRUBIN PROTECTS MURINE

PANCREATIC ISLET CELLS EXPOSED TO HYPOXIC STRESS IN VITRO

Introduction

Type 1 diabetes mellitus (T1DM) is one of the most frequently diagnosed endocrinopathies in dogs, making this species an excellent model for T1DM in humans.65

Successful pancreatic islet transplantation would provide a cure for T1DM in both dogs and humans. Our group has reported successful isolation of canine pancreatic islets from cadaveric donors66, creating a readily available source of islets for transplantation.

However, several hurdles remain before this therapy can be widely applied. Massive cell death due to isolation stress and hypoxia associated with the transplantation process cause loss of up to 70% of functional islets in the first 72 hours after implantation, prior to onset of the acquired immune response.67 Hypoxic stress of islets leads to activation of NF-κB signaling pathways, expression of tissue factor (TF), release of IL-1β from resident macrophages and downregulation of anti-inflammatory cytokines such as IL-10.68 These changes lead to upregulation of cell apoptotic pathways and release of further

31 inflammatory mediators and chemotactic factors from islets, which attract host macrophages. Islet cell necrosis and apoptosis also results in release of intracellular proteins (damage-associated molecular patterns or DAMPs) that trigger an innate host immune response via Toll-like receptor pathways. Cytokine release by host dendritic cells facilitates allorecognition and eventual allograft rejection.69

Bilirubin, a product of the enzyme heme oxygenase (HO-1), is a powerful antioxidant, with anti-inflammatory and cytoprotective properties that have been demonstrated in animal models of ischemia-reperfusion injury.70 Recent work by our group has shown that at therapeutic doses of 10-20µM, bilirubin acts on islet grafts in vitro by suppressing the release of damage associated molecular patterns (DAMPs) and cytokines, resulting in significantly decreased cell death. Administration of bilirubin to murine pancreatic islet allograft recipients at doses of 10-20µM significantly improved graft viability through attenuation of cell apoptosis and reduction of serum inflammatory mediators (IL-1β, TNF-α, sICAM-1, MCP-1).71 In addition, pre-treatment of islet donors with 8.5µM bilirubin also significantly improved islet survival by upregulating expression of protective genes (H0-1, bcl-2) while downregulating pro-inflammatory genes (MCP-1, caspase-3, caspase-8, TNF-α, iNOS) (figure 1).72,73 However, bilirubin is insoluble in water at physiologic pH, is highly protein-bound in plasma and has poor bioavailability, necessitating repeated intravenous or intraperitoneal administration.74 At higher concentrations, bilirubin is well known as a neurotoxin, particularly in susceptible newborns with neonatal jaundice. At concentrations above 25µM, bilirubin has been shown to cross cell membranes and induce immunosuppression and apoptosis via

mitochondrial depolarization.75,76 Thus, this molecule has a relatively narrow therapeutic

range.

Figure 1: The protective effects of bilirubin during pancreatic islet transplant.

Polymeric nanoparticle (NP) drug delivery of hydrophobic compounds can

improve solubility and bioavailability, reducing the effective drug dose and toxicity and

allowing administration via the oral route. Pluronics are amphiphilic, triblock copolymers

that form micelles in an oil-in-water emulsion and Pluronic F127 has been approved by

33 the Food and Drug Administration for use as a pharmaceutical ingredient.22 Chitosan is a natural, biodegradable, cationic polysaccharide, found in the exoskeletons of crustaceans.13 Like Pluronic F127, chitosan is amphiphilic and forms micelles by self- assembly when dispersed in water, with a hydrophobic core that can absorb hydrophobic molecules.77 Its positive surface charge facilitates penetration through cellular membranes, as well as adhesion to mucus glycoproteins.77,78 Pluronic F127-chitosan polymeric NP have been shown to be highly stable and effective in delivery of other hydrophobic molecules.16

We hypothesized that delivery of bilirubin via Pluronic F127-chitosan nanoparticles would allow for sustained drug release and improved drug uptake into pancreatic islet cells in culture when compared to free bilirubin. We further hypothesized that nanoparticle bilirubin (nBR) would improve the viability of isolated murine islets following exposure to hypoxic stress, compared to unencapsulated bilirubin (fBR) or control.

Materials and methods

Materials

Pluronic F127 (MW: 12.6 kDa) was obtained from BASF Corp (Wyandotte, MI,

USA). Chitosan oligosaccharide of pharmaceutical grade (MW: 1.2 kDa, 95.5% deacetylation) was purchased from Zhejiang Golden-Shell Biochemical Co. Ltd

(Zhejiang, China). Bilirubin (bilirubin mixed isomers B4126; Sigma Aldrich, St, Louis,

MO) stock solution of 1.29mM at pH 7.4 was created by the following method: 0.2N

NaOH was added, drop wise, to 0.0584g bilirubin powder. After dissolution of the bilirubin in NaOH, 40mL RPMI (without phenol red) with 10% fetal bovine serum (FBS) was added. The pH was adjusted to 7.4 by gradual addition of HCl. Further RPMI + 10%

FBS was added to yield a total volume of 50mL. The solution was filtered, then the concentration of bilirubin was measured using the colorimetric diazo method (Cobas c311 Analyzer, Roche Diagnostics, Rotkreuz, Switzerland).

Synthesis of Pluronic F127-chitosan nanoparticles

Pluronic F127-chitosan nanoparticles with a core-shell architecture were synthesized using a previously reported method22 with slight modification (figure 2). In short, a total of 30mL Pluronic F127 solution (26mM in benzene) was added drop wise into 30mL 4-nitrophenyl chloroformate (4-NPC) solution (160mM in benzene) and the mixture was stirred for 3 hours in N2 atmosphere at room temperature to activate Pluronic

F127. The activated polymer was then precipitated and filtered in excess (ice-cold) diethyl ether three times and dried under vacuum conditions overnight. To synthesize

Pluronic F127-Chitosan nanoparticles, a total of 500µL of the activated polymer

(300mg/mL or 23.2mM) in dichloromethane was added drop wise into 5mL chitosan solution (15mg/mL or 12.5mM) in deionized (DI) water at pH 10 under sonication using a Branson 450 digital sonifier (Danbury, CT, USA) at 16% of maximum amplitude for 3 minutes. Dichloromethane was then removed by rotary evaporation. The resultant

solution was dialyzed against DI water with a Spectra/Por dialysis tube (MWCO, 50kDa)

overnight and further dialyzed against DI water for 3 hours using 1000kDa Spectra/Por

dialysis tube with a pore size of ~100nm. Consequently, products less than ~100nm were

removed, resulting in a yield of ~18.4% (percentage in weight of final products out of

total reactants used initially). Finally, the sample was freeze-dried for 48 hours to obtain

dry nanoparticles (figure 3).

Figure 2: Procedure for synthesis of Pluronic F127-chitosan NP and for encapsulating a hydrophobic molecule, curcumin.16

Figure 3: A) Scanning electron micrograph of Pluronic F-127 chitosan NP at room temperature; B) Dry nBR at room temperature (feeding ratio 1:20).

Encapsulation of bilirubin to obtain nanoparticle-encapsulated bilirubin (nBR)

Bilirubin was encapsulated in the Pluronic F127-chitosan nanoparticles by adding 20mL bilirubin solution in chloroform (0.25mg/mL) drop wise to 10mL aqueous solution (10mg/mL) of Pluronic-chitosan nanoparticles in DI water under constant stirring. After 1 hour equilibration, the sample was connected to a vacuum system to remove chloroform slowly over 6-8 hours. Then the sample was transferred into a round flask to remove the last traces of chloroform by rotary evaporation. The sample was then filtered through a 0.45µm filter at room temperature and lyophilized for 24 hours to obtain dry nBR that were either used immediately or stored at -20°C for future use. 37

Spectrophotometric analysis of bilirubin content within nanoparticles

Bilirubin is a bile pigment that exhibits powerful autofluorescence, with maximal spectral absorption at a wavelength of 440nm when in aqueous solution and 480nm when bound to bovine serum albumin (BSA).79,80 To determine the mg dose of nBR required for the ensuing experiments, 1mg/mL nBR in PBS was compared to a standard curve of known bilirubin concentrations via spectrophotometric analysis at a wavelength of

440nm. 1mg/mL nBR in PBS was found to have a bilirubin concentration of 4.8µM.

Further calculations of the dose of nBR or eNP were performed based on weight, with the assumption that 1mg nBR and eNP contained equivalent numbers of nanoparticles.

In vitro release studies

Aliquots of 1mL of ~20µM fBR or nBR were suspended within a 20kD

Spectra/Por dialysis membrane, in 25mL of either phosphate-buffered saline (PBS) or

PBS containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The pore size of the dialysis membrane was selected based on the relative sizes of unconjugated bilirubin molecules (0.585kDa), bovine serum albumin (66.5kDa) and radius of the NP

(13.5nm at 37oC). Pores of 20kDa would allow diffusion of spherical molecules with a radius of 1.78nm or smaller.81 Three samples were tested for each of the four groups.

Samples were incubated at 37oC under laminar flow conditions, continuously agitated with magnetic stirrer bars and protected from light (figure 4). 500µL samples of dialysate

38 media were taken at pre-determined time points for a period of 3 days and dialysate was replaced with an equal volume each time. Samples were frozen at -20oC, then spectrophotometric analysis was performed at 460nm, relative to a standard curve of known bilirubin concentrations. Results were expressed as % release compared to initial concentration of bilirubin within the dialysis membrane.

Figure 4: Procedure for testing the release characteristics of nBR and fBR in solutions with or without protein.

Cellular uptake and intracellular distribution of nBR in INS-R3 cells

Murine INS-R3 cells, an insulinoma cell line, were used as a model of pancreatic beta cells. Following cell culture to confluence, cells were seeded into 35mm petri dishes 39 at 1.5 × 105 cells/dish with 1mL RPMI cell culture medium. Each petri dish also contained two 12mm type I collagen-coated glass cover slips, to allow cell adherence.

Glass coverslips were dipped in type I collagen solution (1 mg/mL) in phosphate buffered saline (PBS, 1x by default) for 1 minute and then drying in cell culture hood for 15 minutes in air at room temperature before experiments.

Following overnight incubation at 37oC, cells were further treated with RPMI cell culture media containing 0, 5, 10 or 20µM nBR, fBR or eNP (empty NP), as well as

75nM LysoTracker Red DND-99 (Life Technologies, Grand Island, NY) to fluorescently label acidic organelles (lysosomes). After 1 hour incubation at 37°C, the cells were washed twice with 37°C PBS and fixed with 4% paraformaldehyde at 37°C for 10 minutes. The fixed cells were then incubated with Hoechst 33342 (5µM in PBS) for 10 minutes at 37°C and washed twice with 37°C PBS to stain their nuclei. Finally, the coverslips with attached cells were mounted onto glass slides with Vectashield anti-fade mounting medium (Vector Laboratories, Burlingame, CA) for further examination. The intracellular bilirubin uptake by INS-R3 cells and distribution of bilirubin within the cells was studied qualitatively using Zeiss (Oberkochen, Germany) Apotome (confocal-like) structured illumination microscopy (SIM). Bilirubin demonstrates fluorescence emission at a wavelength of 534nm when associated with plasma membranes.82 Thus, intracellular bilirubin can be detected using a green excitation fluorescence filter, which covers a wavelength of 510-560nm.

Effects of nBR on viability of INS-R3 cells exposed to hypoxic stress

Further INS-R3 cells were then cultured until a monolayer had formed, then were seeded into 24 well cell culture plates at a density of 5x104 cells per well in 1mL cell culture media per well. Media was treated with nBR, fBR or eNP at concentrations of 0-

20µM (two wells per treatment dose). Cells were acclimatized for 4 hours under standard

o incubator conditions (21% O2 at 37 C), then exposed to 8 hours hypoxia (1% O2), followed by 12 hours recovery (standard conditions). MTT viability assays were performed as follows: 50mg MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was dissolved in RPMI cell culture solution (without phenol red), to create

MTT stock solution. 100µL MTT stock solution was added to each well and samples were incubated for 1h at 37oC. Media was aspirated from the wells, cells were rinsed in

1mL PBS (1x) and 0.5mL dimethyl sulfoxide (DMSO) was added to each well. Aliquots of 200µL of the reaction product were transferred to a 96 well plate (2 samples per well of the 24 well plate) and absorbance was measured via spectrophotometry at a wavelength of 562nm. Cell viability was expressed as percentage absorbance of control

(untreated) cells.

Effects of nBR on viability of murine islets exposed to hypoxic stress

Experiments were approved by the Institutional Animal Care and Use Committee.

To assess the effects of nBR, fBR and eNP treatment on viability of murine pancreatic islet cells in vitro, mouse pancreatic islets were isolated from female C57BL/6 donors

(Harlan laboratories, Indianapolis, IN) and maintained in culture using the techniques

41 described by Zmuda et al.83 Following 24h incubation at 37oC, islets were transferred to

35mm petri dishes at a density of ~60 islets per dish and suspended in 1mL media containing either nBR, fBR or eNP at concentrations of 0, 5, 10 or 20µM. Cells were

o incubated under standard conditions (37 C, 21% O2) for 4 hours, then exposed to 24

o hours of hypoxia (1% O2 at 37 C), followed by 12 hours of recovery under standard culture conditions. Media was changed for fresh media containing equivalent drug concentrations prior to recovery. The duration of hypoxia was based on previous work in our laboratory, with the goal of achieving 30-40% cell death in order to demonstrate both potential detrimental and therapeutic effects of NP administration. Islets were hand- picked and transferred to new 35mm petri dishes containing Propidium Iodide and 5µM

Hoescht 33342 in PBS. Cells were imaged using DAPI (blue) and Texas Red (red) channels via epifluorescent microscopy. Images were analyzed using NIH Image J software and the percentage of PI positive cells, indicating % cell death present in each islet, was calculated using a custom islet macro as previously described.84

Statistical analyses

Statistical analyses for release data were performed using MedCalc for Windows, version 12.5 (MedCalc Software, Ostend, Belgium). Area under the curve (AUC) was analyzed using the Kruskall-Wallis non-parametric test with Conover post-tests.

Following outlier detection using Grubbs double sided and Tukey tests, the analysis was re-run with outliers excluded and the results were unchanged. Cell viability data was analyzed using IBM SPSS Statistics for Mac, Version 21.0 (Armonk, NY: IBM Corp).

Shapiro-Wilk and Levene tests were used to test data for normal distribution and homoscedasticity, respectively. For INS-R3 cell viability data, two-factor, univariate analysis of variance (ANOVA) was performed and Tukey HSD post hoc tests were used to compare effects between groups. For murine islet cell viability data, the main effects of

‘treatment group’ and ‘concentration’ on the dependent variable ‘percentage cell death’ were evaluated with the linear mixed procedure. When a significant main effect was identified, a multiple comparison test (Sidak) was performed. Statistical significance was set at p≤0.05.

Results

Physiochemical characterization of nanoparticles

The morphology, size and zeta potential of Pluronic F127–chitosan nanoparticles were determined using the techniques reported by Rao et al.16 Briefly, transmission electron microscopy and a carbon film-coated copper TEM grid were used to visualize NP size, while surface zeta potential of nanomaterials (i.e. NP and nBR) were assessed using a

Brookhaven (Holtsville, NY, USA) 90 Plus/BI-MAS dynamic light scattering (DLS) instrument. Results are presented in table 1.

NP NP Bilirubin: NP Zeta NP Zeta NP type EE LC diameter, diameter, NP ratio potential, potential, (%) (%) nm at nm at (w:w) mV at 22oC mV at 37oC 22oC 37oC

10.1 ± 0.6 ± 355.0 ± nBR 1 : 20 27.1 ± 1.4 4.4 ± 0.4 19.1 ± 4.9 0.2 0.1 9.3

293.7 ± eNP N/A N/A N/A 19.0 ± 1.9 9.3 ± 0.6 20.7 ± 0.7 47.7

Bilirubin release characteristics

Area under the curve for bilirubin release in each of the four groups was significantly

different (p=0.0156). Release was significantly greater for nBR than for fBR and in

solutions containing protein compared to PBS alone (p<0.001). For nBR in PBS +10%

FBS, there was an initial burst of release over approximately 8 hours, followed by a more

steady release up to 48 hours. The initial release burst was not as marked for the fBR

group, but maximal bilirubin release occurred over the first ~48 hours, followed by a

plateau of the release curve (figure 5).

(hours)

Cellular uptake of bilirubin in INS-R3 cells

Qualitatively, INS-R3 cells showed increased uptake of nBR compared to fBR, which was dose-dependent. Uptake of eNP was not visualized, because bilirubin autofluorescence was not present. In cells treated with 10-20µM nBR, colocalization of nanoparticles within acidic organelles (endo-lysosomes) was demonstrated as overlay of the LysoTracker Red and bilirubin (green) fluorescence, resulting in a cumulative yellow color (figure 6).64,85

C Figure 6: Uptake of nBR (bottom row of each image), fBR (middle row) and eNP (top row), at concentrations of 5µM (A), 10µM (B) and 20µM (C), by INS-R3 cells in culture. 46

Effects of nBR on INS-R3 cells under hypoxic conditions

In INS-R3 cells exposed to hypoxic stress, treatment group and concentration had

significant effects on percentage cell death (p<0.001), and there was a significant

interaction between the two factors (p=0.014). Cell viability data is expressed as mean

cell viability, as a percentage of viability of untreated cells, ± standard deviation. Overall,

untreated cells survived significantly better than all other groups (p<0.005). Cells treated

with 5µM nBR (113.2% ± 3.9), had the best viability overall (figure 7).

Figure 7: Mean viability ± SD (expressed as % of viability of untreated cells) of INS-R3 cells treated with 0-20µM nBR, fBR or eNP and exposed to 8h hypoxia.

Effects of nBR on murine islets under hypoxic conditions

In murine islets exposed to hypoxic stress, treatment group (p=0.05) and concentration (p<0.001) had significant effects on percentage cell death. Interaction between the two factors was not significant (p=0.344). Cell viability data is expressed as mean cell death (%) ± standard deviation. Overall, islets treated with nBR (24.7% ± 7.5) survived significantly better than those treated with fBR (43.9% ± 23.4; p=0.047) or eNP

(42.1% ± 21.0) (figure 8). Islets treated with 5µM nBR (18.5% ± 14.1) or 10µM nBR

(25.7% ± 15.7) survived better than untreated islets (33.5% ± 17.5%). Doses of 20µM in every group were significantly more toxic to islets than doses of 5-10µM (p≤0.007).

Following hypoxia, the percentage remaining intact islets in each group were counted and the number compared to the initial count (60 islets per petri dish). Islets treated with 5µM nBR (95.0% intact) and 10µM fBR (76.7% intact) survived better than control (30% intact) (figure 9).

When images of stained islets were analyzed quantitatively (figure 10), central necrosis of islets was seen in untreated groups, as well as those treated with fBR at concentrations of 10-20µM. Central necrosis is a feature of both rat and human islets following hypoxia. Apoptosis and necrosis are particularly pronounced in central areas of large islets.86

Figure 8: Mean cell death ± SD (expressed as % PI positive cells) of murine islets treated with 0-20µM nBR, fBR or eNP and exposed to 24h hypoxia.

Figure 9: Islet survival (expressed as % of initial number of islets) after treatment with 0-20 µM nBR, fBR or eNP and exposure to 24h hypoxia.

Discussion

Release characteristics of nBR and fBR

The release kinetics of entrapped drug from polymeric micelles into the dialysis chamber are affected by diffusion of drug and polymer degradation - Pluronic F127 polymers undergo degradation by hydrolysis in PBS solution.1,87Using the dynamic dialysis method as in this study, the apparent release rate is the net result of drug transport across two barriers in series: release from the nanoparticles into the dialysis chamber followed by diffusion across the dialysis membrane.88

Previous studies of drug release from Pluronic F127-chitosan NPs suspended in

PBS report an initial release ‘burst’ over 8 hours, followed by sustained drug release over the ensuing 48 hours, with plateau of the release curve after 48 hours.21,64 However, only one study21 used methanol extraction of intact NP at sequential time intervals from aqueous NP solutions to accurately determine percent release. This is the first study to use protein in the dialysate solution in an attempt to better reproduce the in vivo release kinetics. In the current study, significantly greater drug release was seen in solutions containing albumin (FBS), compared to PBS alone, which is likely related to the high affinity of bilirubin for albumin in aqueous solution.

Unconjugated bilirubin has poor solubility at pH 7.4 (~70nM), but this is improved in plasma, where 99% of bilirubin (IXα) is bound to albumin in human adults.74 Human serum albumin (HSA) has one high-affinity binding site – an L-shaped

‘pocket’ in subdomain IB – and one or two secondary binding sites with much lower

51 affinity for bilirubin.89 Bovine serum albumin (BSA) has a molecular weight almost identical to HSA (66.5kDa) and the stoichiometry of bilirubin binding to BSA is thought to be comparable to HSA, with one high-affinity and two low-affinity binding sites.80

Bilirubin, complexed to albumin, may have been more stable in solution and diffused more readily across the dialysis membrane than in solutions of PBS alone, where the hydrophobic bilirubin may have precipitated.

Another unexpected finding in the release study was the relatively slower diffusion of fBR across the dialysis membrane compared to nanoencapsulated bilirubin.

Since nBR first had to dissociate from NP, then diffuse across the membrane, slower drug release from the nBR group would have been expected. However, previous studies citing sustained release of drug from NP did not report parallel release data for diffusion of free drug across the same membrane.

A recent study88 questioned the validity of dynamic dialysis as a method for accurately determining drug release from NP in vitro. They proposed that the dual barrier

(release from NP, followed by diffusion across a membrane) complicates data interpretation and may lead to incorrect assumptions about sustained release from NP carriers, since reversible binding of drug to NP within the dialysis membrane determines the drug concentration available for diffusion. They recommended validation of drug release kinetics at varying nanoparticle concentrations and the determination of membrane binding coefficients along with appropriate mechanism-based mathematical modeling to ensure the reliability and proper interpretation of the data.88

Further investigation of drug release kinetics, using ultracentrifugation and

52 ultrafiltration to separate NP from free drug at each time point in the study, as well as continuing the release study over a longer time period, are required to more accurately compare release of nBR to diffusion of fBR..

Improved uptake of nBR by INS-R3 cells

In the current study, nanoparticle delivery of bilirubin achieved a protective effect in isolated murine islets, following hypoxic stress. The mechanism of the improved effects of nBR over fBR is likely the improved, selective uptake of nBR by pancreatic islet cells. Cellular uptake is influenced by NP size and zeta potential. Smaller NPs have a larger surface area, leading to greater cellular uptake and faster release of drug. The nBR in this study, with a diameter 27.1nm at 37oC, were within the ideal range for uptake via endocytosis of 10-30nm.2 In addition, the positive surface charge of nBR at 37oC

(19.1 +/- 4.1mV) likely facilitated its uptake by mammalian cells, whose plasma membrane is usually negatively charged.16

Pancreatic beta cells are highly active metabolically, especially under conditions of increased glucose. The stimulation of insulin release by glucose is accompanied by an enhanced uptake of macromolecules, via a specific exocytosis-endocytosis coupling mechanism.90 However, the cellular entry of macromolecules like bilirubin, via simple diffusion, is impaired by the poor permeability and selectivity of cell membranes and by degradation within lysosomes following internalization by endocytosis. Polymeric nanoparticles are capable of escaping degradation within the endo-lysosomal compartment, resulting in more efficient intracellular delivery.91 53

LysoTracker Red is a fluorescent marker for secondary endosomes and lysosomes, which is colorless at physiologic pH, but has red fluorescence at the acidic pH of 4-5 of endosomal compartments. In the current study, when nBR (green fluorescence) was colocalized within the endo-lysosomal compartment with LysoTracker Red, yellow fluorescence resulted. However, at 1 hour post-incubation, some nBR was also localized in the cytoplasmic compartment, as seen from the appearance of green fluorescence of

NPs (figure 6). This is consistent with the findings of previous studies of Pluronic F127- chitosan NP and it can be inferred that NPs were probably escaping rapidly from the endo-lysosomal compartments into the cytoplasmic compartment following their uptake.22,91 These observations indicate that endocytosis via the endosome/lysosome system is an important mechanism for cellular uptake of the Pluronic F127-chitosan nBR.

Effect of nBR on INS-R3 cells exposed to hypoxic stress

This study showed a trend towards a protective effect of 5µM nBR in insulinoma cell line (INS-R3) cells exposed to hypoxic stress (figure 7). However, untreated cells still survived better than any other group, overall. Since the MTT assay provides cell viability data relative to control cells, rather than as an absolute value, it is not possible to compare % cell death overall between INS-R3 and murine islet cells following hypoxia.

However, it is possible that the cancer cell line was less susceptible to the detrimental effects of hypoxia than pancreatic islets, which may have resulted in attenuation of the protective effects of NP. In a previous study characterizing cell death in murine islets and

MIN6 insulinoma cell line cells exposed to transplant stressors in vitro, murine islets 54 were found to exhibit marked plasticity in cell death modes, in contrast to MIN6 cells, which died almost exclusively via classical apoptosis.92 These observations suggest that insulinoma cell lines may not be an appropriate or representative model for murine pancreatic islets in future experiments. Another explanation for the modest protective effects of nBR in INS-R3 cells is that neoplastic cells tend to accumulate larger numbers of NP through endocytosis, due to their increased metabolic activity. This may have resulted in a relative overdose of NP polymers, overwhelming any protective effects of nBR.

Protective effects of nBR on murine islets following hypoxic stress

Doses of 5-10µM nBR were protective of murine pancreatic islets following hypoxic stress, in agreement with previous studies, which reported therapeutic doses of fBR of 8.5-10µM.72,73 In general, the dosing efficiency of nano-encapsulated drug is expected to be greater than free drug, due to sustained drug release combined with rapid, preferential uptake by metabolically active cells.63 Targeted drug delivery to islets, using an islet-specific ligand, such as an ‘islet-homing peptide’64, may further increase the efficiency of nBR and reduce the therapeutic dose.

Treatment of islets with nBR prevented central necrosis of islets when PI images were assessed quantitatively (figure 10). Untreated islets and those treated with >5µM fBR exhibited characteristic central necrosis, while islets treated with 5µM nBR did not.

Giuliani et al86 investigated the relative effects of hypoxia (1% oxygen for 24 hours) and nutrient deprivation on pancreatic islets and MIN6 insulinoma cells and concluded that 55 the gradual transition from an apoptotic to a necrotic morphology in the central cells of hypoxic pancreatic islets is the result of the combined effects of hypoxia and nutrient or serum deprivation, most likely due to impaired diffusion. The mechanism by which nBR protects against central necrosis may be improved uptake of nBR by central islet cells, compared to fBR, since uptake of nBR appears to be via active transport, rather than simple diffusion as for fBR. Previous work in our laboratory investigating the protective effects of fBR on isolated murine islets exposed cells to 3 hours hypoxia found significant improvements in cell survival. However, exposure to 24 hours hypoxia may have overwhelmed the protective effects of fBR, and highlights the improved effectiveness of NP drug encapsulation. This may have particular clinical importance in canine islet transplantation, since both murine and canine islets are composed primarily of β-cells clustered in a central core, surrounded by smaller numbers of α-cells, δ-cells,

PP-cells and ε-cells in the periphery. This is in contrast to human islets, which have fewer

β-cells, randomly distributed throughout the islet.93

Toxicity of eNP to murine islets and INS-R3 cells

Interestingly, eNP were found to be toxic to both INS-R3 cells and murine islets, following hypoxic stress. Treatment of murine islets with eNP at 5-10µM resulted in a higher rate of cell death (measured via PI staining) and a lower number of intact islets than treatment with nBR at the same doses. Murine islets and INS-R3 cells in this experiment were treated with 1-4mg/mL nBR or eNP, in order to achieve bilirubin concentrations of 5-20µM. Given the low loading content of the nBR (0.6% by weight of

56 each NP), toxicity may have resulted from the relatively higher dose of NP needed to achieve therapeutic doses of BR.

Some previous studies of the effects of Pluronic F127-chitosan NP on both cancerous and non-cancerous cultured cell lines in vitro have found low toxicity (>95% viability) at doses of 1-10mg/mL.21,22 In contrast, a study investigating PLGA-PEG NP for delivery of an immunosuppressive drug to murine islet capillary endothelial cells in vitro, found a dose-dependent pro-inflammatory effect of empty NP, at doses of 10-

50µg/mL.64 Although chitosan is considered non-toxic and has been approved by the

FDA for wound dressings, it has been shown to have antitumor effects on numerous human cancer cell lines in vitro.77 Its mechanism of action is to induce a robust IL-1β response, via K+ efflux, formation of reactive oxygen species and lysosomal destabilization.94 The mechanism by which NP cause dose-dependent cytotoxicity of both

INS-R3 cells and murine islets is not clear from this study; further investigation in this area is warranted.

Negligible protective effects and dose-dependent toxicity of fBR

In contrast to previous studies, treatment with fBR had negligible protective effect on murine islets and dose-dependent cytotoxicity was evident. Doses of 25-50µM bilirubin have been found to interfere with mitochondrial respiration and induce necrosis and apoptosis of cells in culture.76 The cytotoxicity of nBR at high doses in this study is therefore likely a result of the additive toxicity of bilirubin and the NP themselves.

Although a larger number of intact islets were present following treatment with 10µM 57 fBR than control, PI viability staining showed a higher percentage cell death in this group.

Limitations and future directions

This study has several important limitations. The in vitro nature of the experiments may not accurately represent the behavior of nBR in an in vivo model of transplant. Small sample sizes, as well as inherent inaccuracies in the use of spectrophotometric and fluorescent imaging analyses may have resulted in type II statistical error. The mechanism by which nBR protects hypoxic murine islets is the subject of future work for our group. Islet function testing, as well as cytokine assays, would help to guide future developments in nanotechnology research for islet transplant.

Future studies should also focus on developing a less cytotoxic vehicle for nBR administration and improving the loading content of nBR.

Conclusion

Pluronic F127-chitosan nanoencapsulation of bilirubin is feasible and results in improved cellular uptake via endocytosis in INS-R3 cells. In this in vitro investigation, dose-dependent protective effects of nBR on hypoxic murine islets were demonstrated, with doses of 5µM nBR providing maximum protective benefit. Meanwhile, empty NP exhibited dose-dependent cytotoxicity, and the previously reported protective effects of

58 fBR administration were apparently overwhelmed by the prolonged period of hypoxia.

Treatment with nBR resulted in preservation of normal islet architecture, as well as prevention of central necrosis, at a dose of 5µM. This study identifies a need for more accurate methods of determining release kinetics of nanoencapsulated hydrophobic molecules. It also draws into question the appropriateness of insulinoma cell lines as a model for isolated pancreatic islets in vitro. More efficient loading of bilirubin into NP, the mechanisms by which nBR exerts its protective effects, and the development of less cytotoxic NP for delivery of cytoprotective agents to transplanted cells should be the subjects of future research.

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