Enhanced Delivery of Gold Nanoparticles with Therapeutic Potential for Targeting Human Brain Tumors

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1 Enhanced Delivery of Gold Nanoparticles with Therapeutic Potential for Targeting Human Brain Tumors by Arnold B. Etame A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy, Graduate Department of Laboratory Medicine and Pathobiology University of Toronto Copyright by Arnold B. Etame, 2012

2 ii Enhanced Delivery of Gold Nanoparticles with Therapeutic Potential for Targeting Human Brain Tumors Arnold B. Etame Doctor of Philosophy Laboratory Medicine and Pathobiology University of Toronto 2012 Abstract The blood brain barrier (BBB) remains a major challenge to the advancement and application of systemic anti- cancer therapeutics into the central nervous system. The structural and physiological delivery constraints of the BBB significantly limit the effectiveness of conventional chemotherapy, thereby making systemic administration a non- viable option for the vast majority of chemotherapy agents. Furthermore, the lack of specificity of conventional systemic chemotherapy when applied towards malignant brain tumors remains a major shortcoming. Hence novel therapeutic strategies that focus both on targeted and enhanced delivery across the BBB are warranted. In recent years nanoparticles (NPs) have emerged as attractive vehicles for efficient delivery of targeted anti- cancer therapeutics. In particular, gold nanoparticles (AuNPs) have gained ii

3 iii prominence in several targeting applications involving systemic cancers. Their enhanced permeation and retention within permissive tumor microvasculature provide a selective advantage for targeting. Malignant brain tumors also exhibit transport- permissive microvasculature secondary to blood brain barrier disruption. Hence AuNPs may have potential relevance for brain tumor targeting. However, the permeation of AuNPs across the BBB has not been well characterized, and hence is a potential limitation for successful application of AuNP- based therapeutics within the central nervous system (CNS). In this dissertation, we designed and characterized AuNPs and assessed the role of polyethylene glycol (PEG) on the physical and biological properties of AuNPs. We established a size- dependent permeation profile with respect to core size as well as PEG length when AuNPs were assessed through a transport- permissive in-vitro BBB. This study was the first of its kind to systematically examine the influence of design on permeation of AuNPs through transport- permissive BBB. Given the significant delivery limitations through the non- transport permissive and intact BBB, we also assessed the role of magnetic resonance imaging (MRI) guided focused ultrasound (MRgFUS) disruption of the BBB in enhancing permeation of AuNPs across the intact BBB and tumor BBB in vivo. MRgFUS is a novel technique that can transiently increase BBB permeability thereby allowing delivery of therapeutics into the CNS. We demonstrated enhanced delivery of AuNPs with therapeutic potential into the CNS via MRgFUS. Our study was the first to establish a definitive role for MRgFUS in delivering AuNPs into the CNS. In summary, this thesis describes results from a series of research projects that have contributed to our understanding of the influence of design features on AuNP permeation through the BBB and also the potential role of MRgFUS in AuNP permeation across the BBB. iii

4 iv Acknowledgments This thesis would not have been possible without the support and efforts of multiple individuals. A profound gratitude to my PhD supervisor, Dr. James Rutka who epitomizes the core ideals of a dedicated surgeon scientist, compassionate physician and excellent teacher. The limitless resources and guidance provided by Dr. Rutka have been extremely instrumental in making this research experience a success and it is privilege to have been part of the lab. I am also thankful for the guidance and mentorship Dr. Warren Chan has provided throughout my research training, broadening my horizons on nanotechnology, and for serving on my thesis supervisory committee. I am particularly grateful to all members of the Chan Lab especially Dr. Steve Perrault who introduced me to the design and characterization of nanomaterials. I am also thankful for the guidance and mentorship Dr. Annie Huang has provided throughout my research training, and for her instrumental role in serving as the chair of my thesis supervisory committee. I am very grateful to Dr. Kullervo Hynynen for graciously collaborating on the focused ultrasound projects and also to the members of the Hynynen Lab for their continued guidance and assistance. I am very thankful to both current and past members of the Rutka lab including Dr. Christian Smith, Dr. Roberto Diaz, Dr. Adrienne Weeks, Dr. Paul Kongkham, Dr Yuzo Terakawa, Dr. Claudia Faria, Mr. Brian Golburn, Mr. Jim Loukides, Ms. Sara Onvani, and Mr. Andres Restrepo. In particularly, I am most grateful to Roberto with whom I worked very closely on focused ultrasound studies. I am equally very grateful to Christian for his valuable suggestions and guidance. I am also very thankful to my neurosurgery colleagues at the University of Michigan for their encouragement and support. Lastly, I would like to thank my family and friends whose continued support has been unprecedented. iv

5 v Table of Contents Acknowledgments...iv Table of Contents...v List of Tables...ix List of Figures...x List of Figures...xi List of Abbreviations... xii List of Abbreviations...xiii Chapter 1 INTRODUCTION CENTRAL PROBLEM NANOTECHNOLOGY AND ONCOLOGY GENERAL OVERVIEW DESIGN CONSIDERATIONS FOR NANOMATERIALS REPRESENTATIVE NANO-CARRIERS FOR ONCOLOGY GOLD NANOPARTICLES AS PROTOTYPE ANTI-CANCER DELIVERY SYSTEMS OVERVIEW AND SYNTHESIS APPLICATIONS OF GOLD NANOPARTICLES IN ONCOLOGY BLOOD BRAIN BARRIER (BBB) CHALLENGES FOR GOLD NANOPARTICLE DELIVERY PLATFORMS STRATEGIES TO ENHANCE PASSIVE BBB PERMEATION SCOPE OF DISSERTATION...24 Chapter FEASIBLE SYNTHESIS AND CONJUGATION OF GOLD NANOPARTICLES ABSTRACT INTRODUCTION...27 v

6 vi 2.3 METHODS AuNP Synthesis Scheme AuNP Characterization Conjugation of PEG onto AuNP Conjugation of IL13Ra2 Antibody onto AuNP and Uptake in U251 Glioma Cell Lines Conjugation of sirna onto AuNP Intracellular delivery of sirna-aunp Conjugates in GFP-expressing U287 glioma cell lines RESULTS AuNP Synthesis AuNP Characterization Conjugation of PEG onto AuNP Conjugation of IL13RA2 Antibody onto AuNP and Uptake in U251 Glioma Cell Lines Conjugation of sirna onto AuNP Intracellular delivery of sirna-aunp conjugates in green fluorescent protein (GFP) -expressing U251 glioma cell lines DISCUSSION SIGNIFICANCE...53 Chapter DESIGN AND POTENTIAL APPLICATION OF PEGYLATED GOLD NANOPARTICLES WITH SIZE-DEPENDENT PERMEATION THROUGH BRAIN MICROVASCULATURE: A BBB TRANSPORT-PERMISSIVE MODEL ABSTRACT INTRODUCTION METHODS Materials & Cell Culture...58 vi

7 vii AuNP Synthesis AuNP Characterization AuNP PEGylation Construction of an In-vitro Brain Microvasculature Permeation Model Assessment of PEG-AuNP Permeation Statistical Analysis RESULTS Synthesis and Characterization of AuNP Design and Characterization of PEG-AuNP In-Vitro Brain Microvasculature Permeation of PEG-AuNP DISCUSSION SIGNIFICANCE...79 Chapter ENHANCED DELIVERY OF GOLD NANOPARTICLES WITH THERAPEUTIC POTENTIAL INTO THE BRAIN USING MRI-GUIDED FOCUSED ULTRASOUND IN A PRECLINICAL ANIMAL MODEL ABSTRACT INTRODUCTION BACKGROUND FUS DISRUPTION OF BBB METHODS Characterization of AuNP MRgFUS Delivery Scheme Experimental Design Histology and Silver-Augmentation for in-situ AuNP Detection Measurement of Brain and Organ Biodistribution of AuNP...93 vii

8 viii Statistical Analysis RESULTS Characterization of AuNP MRgFUS Disruption of BBB Gross Pathological Examination of the Brain Histological Examination of The Brain Histological Examination of The Liver, Spleen, and Kidney Qualitative Assessment of AuNP Content Within The Brain Biodistribution of AuNP Outside the CNS DISCUSSION SIGNIFICANCE Chapter CONCLUSIONS AND FUTURE DIRECTIONS STATEMENT OF MAJOR CONCLUSIONS AuNP SYNTHESIS AND CONJUGATION FUTURE DIRECTIONS AuNP BRAIN MICROVASCULATURE PERMEATION FUTURE DIRECTIONS FUS-MEDIATED AuNP DELIVERY IN CNS FUTURE WORK CONCLUSION Bibliography viii

9 ix List of Tables Table 1.1 Enhanced BBB delivery 23 Table 2.1. Absorbance and HD ranges for from various reducing agents 37 Table 2.2. Absorbance and HD changes with AuNP PEGylation 41 Table 3.1. TEM cores sizes and hydrodynamic diameter 63 Table 3.2. Permeation data for PEG coated AuNP 72 Table 3.3. Statistical significance between PEG-AuNP permeation 73 Table 4.1. Advantages of FUS-mediated therapeutic delivery 87 Table 4.1. Enhanced CNS biodistribution of AuNP with 2hr 105 Table 4.2. Biodistribution of AuNP outside the 2hr 107 ix

10 x List of Figures Figure 1.1 Distribution of All Primary Brain and CNS Tumors 2 Figure 1.2 Distribution of All Primary Brain and CNS Gliomas 2 Figure 2.1 Influence of reducing agent strength on AuNP Size 36 Figure 2.2 Representative UV-VIS Spectra of AuNP 38 Figure 2.3 Representative TEM evaluation of AuNP morphology 39 Figure 2.4 UV-VIS Spectra of PEG-modified AuNP 41 Figure 2.5 Electrophoretic mobility of AuNP 42 Figure 2.6 Surface binding and uptake of AuNP conjugates 44 Figure 2.7 Electrophoretic retardation analysis of sirna binding. 45 Figure 2.8 TEM internalization of various AuNP conjugates 47 Figure 2.9 Internalization of GFP-targeting sirna-aunp conjugates 48 Figure 3.1 Experimental scheme for BBB permeation 60 Figure 3.2 UV-VIS Spectrocopy Characterization of AuNP 63 Figure 3.3 TEM Characterization of AuNP 64 Figure 3.4 Absorption Spectra of PEG coated AuNP 65 Figure 3.5 Effects of PEG surface chemistry on zeta potential 66 Figure 3.6 Effects of PEG surface chemistry on HD 67 Figure 3.7 Aggregation of AuNP in the absence of PEG 68 x

11 xi List of Figures Figure 3.8 PEG surface chemistry confers stability 69 Figure 3.9 Permissive transport of PEG-AuNP across BBB 70 Figure 3.10 Kinetics of PEG-AuNP permeation across BBB 75 Figure 3.11 Model of PEG-AuNP BBB permeation 80 Figure 4.1 Schematic of enhanced BBB delivery following FUS 87 Figure 4.2 Preclinical FUS BBB disruption system 88 Figure 4.3 Characterization of AuNP 94 Figure 4.4 MRI demonstrating disruption of BBB by MRgFUS 96 Figure 4.5 Brain histopathology after BBB disruption by MRgFUS 98 Figure 4.6 CNS localization of AuNP after BBB disruption by MRIgFUS 100 Figure 4.7 Brain histology in the subacute phase after MRIgFUS 102 Figure 4.8 Spleen, liver and kidney histology after MRIgFUS 104 Figure 4.9 Enhanced CNS delivery of AuNP after MRIgFUS 106 Figure 4.10 Biodistribution of AuNP outside the CNS after MRgFUS 107 Figure 4.11 MR imaging of MRgFUS of a glioma graft 114 Figure 4.13 MRgFUS-mediated delivery of AuNP to glioma graft 115 Figure 4.13 AuNP localization in rat glioma without MRgFUS 116 Figure 4.14 MRgFUS model system for CNS AuNP delivery 117 xi

12 xii List of Abbreviations AuNPs CNS DLS EPR EGFR FUS GFP HD HPMA HAuCl4 FUS H2O ICP-AES ICP-MS IL13R2A IL13Ra2-AuNP IL13Ra2-PEG MRgFUS MRI MW NaBH4 NIR Gold nanoparticles Central nervous system Dynamic light scattering Enhanced permeability and retention Epidermal growth factor receptor Focused ultrasound Green fluorescent protein Hydrodynamic diameter N-(2-hydroxylpropyl)methacrylamide Gold chloride Focused ultrasound Water Inductively coupled plasma atomic emission spectroscopy Inductively coupled plasma mass spectroscopy Interleukin-13 Receptor 2-alpha Interleukin-13 Receptor 2-alpha/Gold nanoparticle conjugate Interleukin-13 Receptor 2-alpha/PEG conjugates Magnetic resonance guided focused ultrasound Magnetic resonance imaging Molecular weight Sodium borohydride Near infrared light xii

13 xiii List of Abbreviations NPs PAMAM PEG PEG-AuNP PLA PLGA QD RA RBECs RES sirna sirna-aunp sirna-aunp-il13ra2 sirna-aunp-peg TNF-alpha UV-VIS Nanoparticles Polyamidoamine Polyethylene glycol PEG Gold Nanoparticles Poly(lactic acid) Poly(lactic co-glycolic acid) Quantum dot Rat Astrocytes Rat Brain Endothelial Cells Reticulo-endothelial system Small interfering RNA sirna-gold nanoparticle conjugates sirna-gold nanoparticle-interleukin-13 Receptor 2-alpha sirna-gold nanoparticle-peg Tumor necrosis factor alpha Ultra-violet and visible xiii

14 1 Chapter 1 INTRODUCTION 1.1 CENTRAL PROBLEM According to national statistics, cancer remains a leading cause of death in North America 1. While major strides have been made in the treatment of several systemic cancers, progress is still lagging in the management of malignant brain tumors. The most common primary malignant brain tumor is the malignant astrocytoma, which represents the most common primary brain tumor in adults(figure 1.1 & 1.2) 1, 2. A quintessential pathological feature of the malignant astrocytoma is its propensity towards extensive infiltration and invasion into normal brain parenchyma thereby evading effective targeting. Consequently, the current multimodal therapeutic paradigm of conventional chemotherapy, radiotherapy and surgical resection is largely ineffective against this disease. Not surprisingly, the prognosis for patients afflicted with malignant astrocytomas remains dismal 3-6, with the overwhelming majority of patients dead within 2 years 3. Given the highly invasive nature of the disease, localized therapies such as surgery and radiation therapy are not always adequate for disease eradication or control. Furthermore, conventional chemotherapy has its shortcomings. In essence, with chemotherapy there is a lack of targeting specificity due to the intrinsic biology of the tumor cells. Moreover, there are both structural and physiological delivery limitations imposed by the blood brain barrier (BBB) which significantly compromise the bioavailability of targeting therapeutics within the brain Hence delivery strategies that confer both enhanced tumor- targeting 1

15 2 specificity as well as BBB permeation are highly essential if significant clinical outcomes are to be realized in patients with malignant brain tumors. Figure 1.1: Distribution of All Primary Brain and CNS Tumors by Histology based on The Central Brain Tumor Registry of the United States (CBTRUS) data 2. 2

16 3 Figure 1.2: Distribution of All Primary Brain and CNS Gliomas by Histology based on The Central Brain Tumor Registry of the United States (CBTRUS) 2 data. Recent advances within the field of nanotechnology have created opportunities for novel diagnostic and therapeutic delivery applications in oncology As a result, there are currently several nanoparticle- chemotherapy conjugates in clinical and preclinical trials for systemic cancers 27, 28. Doxil which is a liposomal formulation of doxorubicin 27 and Abraxane, a paclitaxel- bound protein particle 27, 29 are currently employed as first- line treatments in various systemic cancer types. In particular, nanotechnology- based delivery systems are highly attractive since they can circumvent some of the challenges associated with conventional therapy. Within the context of tumor targeting, nanoparticles (NPs) employ a strategy called the enhanced permeability and retention (EPR), which takes advantage of the porous vasculature of solid systemic tumors Via EPR, NPs can easily permeate and accumulate within tumors leading to sustained delivery of cancer therapeutic payloads. The vasculatures of malignant brain tumors similarly demonstrate 3

17 4 porosity leading to a transport- permissive microvasculature secondary to compromise of the BBB Hence there are potential opportunities to extrapolate or translate several of the NP- mediated therapeutic targeting strategies that are currently employed for solid systemic cancers to the management of malignant brain tumors. However, in order to effectively extrapolate systemic applications of NPs into the central nervous system (CNS), a better understanding of design implications on BBB permeation is highly desirable. Design features that enhance permeation through transport- permissive microvasculature can be optimized for passive and active delivery applications for malignant brain tumors. Furthermore, an improved understanding of strategies that could enhance permeation in conditions where the BBB is intact could have significant relevance for targeting tumors cells in areas where the BBB remains intact. Given the rapid advancement of nanotechnology in conjunction with improved understanding of brain tumor biology, it is highly conceivable that nanotechnology will undoubtedly play a central role in the delivery of targeted neuro- oncology therapeutics. 1.2 NANOTECHNOLOGY AND ONCOLOGY GENERAL OVERVIEW Cancer remains a very devastating disease with over 10 million new cases worldwide every year 38. Chemotherapy, which is one of the mainstay treatment modalities for most cancers, is plagued with systemic toxicity and poor targeted delivery efficiency. The quest for targeted delivery as well as passive targeting within porous tumor vasculature led to the emergence of nanomaterials as ideal conduits for delivery of therapeutics in oncology. It was reasoned that through rational design of nanomaterials, tumor- targeting efficiency 4

18 5 could be enhanced thereby improving outcomes while minimizing systemic toxicities. In addition, the unique physical and chemical properties acquired by materials within the nanoscale range afford potential applicability for cancer detection and diagnosis. For instance, nanomaterials such as gold nanoparticles, quantum dots, magnetic nanoparticles, carbon nanotubes and nanowires have been employed for detecting cancer biomarkers 26, 39. Hence nanotechnology was poised to play a significant role in the advancement of cancer therapeutics and diagnosis. Nanotechnology developed as an interdisciplinary endeavor that combined the unique attributes of chemistry, physics, engineering, biology, and medicine. The field generated a significant amount of enthusiasm in light of the great potential for personalized treatment of cancer through enhanced detection and targeted treatment of cancer 40. Nanotechnology has experienced significant growth over the last two decades with realization of a substantial number of potential nanotechnology applications both in the physical and biological sciences. More importantly, the advent of nanometer scale devices has led to the development of novel sophisticated diagnostic and therapeutics tools mostly for potential practicality in nanomedicine 23, Nanomedicine emerged as an amalgamation and incorporation of the unique diagnostic and theranostic potentials of nanotechnology into medicine The goal is to design nano- scale devices for drug delivery, targeting, diagnostic and imaging applications for disease processes throughout the body. Some of the nanotechnology- derived arsenals that have potential nanomedicine utility includes liposomal nanoparticles 48, functionalized nanotubes 49, iron oxide nanoparticles 50, polymeric micelles 51, dendrimers 52, nanoshells 53, 5

19 6 and gold nanoparticles 54. Nanomedicine is a relatively new field with significant growth potential as advances continue to occur in the engineering and design of nanomaterials. Nanomaterials encompass a wide spectrum of engineered products designed as particulate systems. They can be broadly classified as organic or inorganic particulate systems. Organic particulate systems include polymeric NPs, polymer- conjugates, dendrimers, carbon nanotubes, fullerenes and lipid- based NPs. Organic NPs systems are most employed for drug delivery applications. Inorganic particulate systems are largely composed of metallic NPs and semiconductor NPs. Metallic NPs such as gold nanoparticles (AuNPs) have thermal and optical properties that can be exploited for diagnostic and therapeutic cancer applications 55. Other metallic NPs such as iron oxide NPs have magnetic dipoles which is can be exploited for magnetic resonance imaging (MRI) applications. Semiconductor NPs or quantum dots (QDs) are typically engineered from heavy metals such as cadmium and selenium. They have very high optical extinction coefficients, enhanced tunable fluorescence and resistance to photobleach 56. The photo- stability of QDs is a major advantage over commercial fluorophores, which have a propensity to photobleach. diagnostics. Hence QDs have emerged as promising tools for biomedical imaging and However, given that QDs are designed from heavy metals and are not biodegradable, potential toxicity concerns have hampered their clinical applications in vivo. Nanomaterials are traditionally engineered to encompass a diameter range between nanometer (nm) in size. Nanotechnology therefore affords the engineering of structures that are closer to the atomic range but much smaller than bulk molecules. As a consequence of the nanometer size range, nanomaterials have larger surface area per weight ratios than their bulk molecule counterparts, which is very advantageous for 6

20 7 delivery and detecting applications 27. The large surface area allows for incorporation of a high density of biomolecules such as ligands, nucleic acids and antibodies. Moreover, the resulting nanometer size range has significant implications for the physical characteristics as well as biological interactions of NP. Unlike bulk molecules or discreet atoms, engineered nanomaterials exhibit unique size- dependent optical, magnetic, electronic and catalytic features. Metallic NPs such as gold (AuNP) exhibit very strong optical absorbance secondary to intense surface plasmon response 57. Metallic NP also demonstrated size- dependent electric field responses when subjected to an external electromagnetic radiation source 58. Semiconductors nanocrystals or quantum dots (QDs) productive intense emission spectra upon optical excitation that are dependent on particle core- size 59, 60. Furthermore, NPs derived from transition metals acquire surface catalytic properties as well 61. Alterations in the shapes and chemical compositions of nanomaterials can also influence the physical and biological interactions of the nanomaterial. The overarching advantage of nanotechnology is the ability to employ size as a basis for tuning the physical properties of the nanomaterial thereby resulting to a broad range of potential biomedical applications. Nanomaterials are equally attractive since they allow for multi- functionality. Reactive groups such as carboxylates, esters, alcohols or amines can be easily functionalized onto the surface of the nanoparticle thereby facilitating conjugation of targeting biomolecules such as peptides, antibodies and nucleic acid 62. Nanomaterials generally have large surface areas that can accommodate multiple binding ligands thereby allowing multivalent biological interactions for instance with cell surface receptors 63. Given their large packing densities, nanomaterials can augment the multivalent binding 7

21 8 affinities of even low- affinity ligands, which is a significant therapeutic advantage 64, 65. Similarly, the dense packing ratio is favorable for incorporation of multiple therapeutic agents onto the same nanostructure. Nanomaterials have sizes that are comparable to large biological molecules such as enzymes, receptors, and antibodies, which is an inherent advantage. Size similarities allow for cross integration of nanomaterials with biomolecules in vivo. For instance antibodies, peptides and nucleic acids can be easily incorporated onto nanomaterials thereby enhancing delivery capacity into targeted cells. Furthermore, ligands can be incorporated onto nanomaterials in order to elicit a desired signal transduction response upon binding onto the targeted surface receptor. Another potential advantage of the nanoscale size is the potential for nanomaterials to traverse biological membranes for efficient delivery of therapeutics and biological interactions without necessarily compromising the integrity of membranes 66. Nanomaterials can be designed to be both biocompatibility and biodegradable, which in essence broadens their biomedical applications. For instance, lipid- based carriers such as liposomes have become ideal vehicles for therapeutics since they are biocompatible and have highly favorable drug- encapsulation potential 27. Not surprisingly liposomal delivery is currently extensively employed in the delivery of several chemotherapeutic with FDA approval Nanotechnology clearly affords a novel approach for targeted delivery in medicine and cancer in particular. The favorable pharmacokinetics and drug- encapsulation potential allows for maximum desired pharmacologic response with minimal potential toxicity. In 8

22 9 addition nanotechnology may offer a unique advantage over conventional diagnostic assays and imaging modalities DESIGN CONSIDERATIONS FOR NANOMATERIALS Nanoparticle- based drug delivery vectors are unique in light of their ability to passively target tumor vasculature through enhanced permeability and retention (EPR) effect Solid tumors are often characterized by a vasculature that appears to be very porous to macromolecules. The vascular permeability cut- off pores sizes of tumor vessels can range from nm in diameter based on size- dependent assessment of liposomes permeation in human xenografts 70. Given their smaller size- range, therapeutic nanomaterials can therefore easily permeate through leaky tumor vasculature. Furthermore, the absence of a well- organized lymphatic drainage system within solid tumors, facilitates retention of permeated nanomaterials 71. Once resident within the interstitial spaces of the tumor, nanomaterials can either release their therapeutic payload onto the surrounding environment or in most instances will depend on various uptake mechanisms for intracellular delivery applications. In order to optimize the attributes of anti- cancer nanomaterials, several considerations should be taken during the engineering and design process. Size appears to be one of the most important parameters for nanomaterials since size ultimately determines biodistribution and hence fate of the nanomaterial. Most nanomaterials employed for cancer therapeutics are within the <100 nm range given that larger NP have diffusion limitations within the extracellular space 72. One study looking at lipid carriers showed a very favorable peri- tumoral diffusion of neutral to slightly charged particles <100 nm 73. Lastly, data from several studies suggests that that NP close to 50 nm 9

23 10 size range have the best cellular uptake kinetic profile Hence, cancer nano- therapeutics should be designed with the above size considerations in mind. Beyond size, the surface properties of the nanomaterial are very important as well. Nanomaterials have larger surface area- volume ratios, which has implications for their biological interactions. Because of the large surface area, nanomaterials can serve as carrier for much larger drug payloads than other conventional carriers. For instance nanomaterials within the therapeutic range can carry thousands of small- interference RNA (sirna) molecules on their surface 77. Given that the payload is contained within the nanomaterial, the pharmacokinetics of the nanomaterial is not usually affected. Another aspect of surface property to take into consideration is the surface charge. Highly negatively charge nanomaterials could suffer from shorter circulation times secondary to rapid clearance by the reticulo- endothelial systems (RES) within the liver and spleen. Rapid clearance of nanomaterials can be minimized through surface functionalization with PEG, which creates steric hindrance and prevents opsonization by the RES 78. Hence surface properties play a critical role in the biological fate of the nanomaterial. Another factor of consideration is the role of targeting ligands especially when active targeting is desired. Targeting ligands enhance intracellular uptake by tumors cells for therapeutics that require internalization for desired pharmacologic effects. However, the accumulation of nanomaterials within tumor interstitial spaces is largely driven by the EPR effect, which is a form of passive targeting. Taking advantage of the large surface density, nanomaterials can be designed to accommodation a wide variety of targeting ligands thereby improving specificity of drug delivery applications. 10

24 REPRESENTATIVE NANO-CARRIERS FOR ONCOLOGY The interest surge in nanotechnology research has led to development of several nano- carrier based systems for cancer applications. Nanocarriers have been successfully employed for drug delivery as well as cancer targeting applications such as photothermal ablation, imaging, and sentinel lymph- node mapping 25, 26, 79. Representative nanocarriers include polymer- conjugates, polymeric nanoparticles, lipid- based nanoparticles (liposomes and micelles), dendrimers, carbon nanotubes, and metallic nanoparticles such as gold in particular. Polymer- conjugates refer to either drugs or pharmacologic peptides that have been conjugated to polymers in order to enhance delivery via improved pharmacokinetics. Polymer- conjugates have demonstrated clinical utility in the targeting of tumor angiogenesis The most commonly employed polymers include N- (2- hydroxylpropyl)methacrylamide (HPMA) copolymer, poly- L- glutamic acid, poly(ethylene glycol) (PEG), and Dextran 25. These polymers have been used to generate conjugates of chemotherapy agents such as doxorubicin, camptothecin, paclitaxel, and cis- platinum 25. Several PEGylated nano- based protein therapeutics have been used in clinical trials. The advantages of PEGylation include increase protein solubility, decreased immunogenicity, increased circulation times and decreased renal 78. Representative examples included PEG- l- asparaginase for acute lymphoblastic leukemia 84 and PEGylated interferon for melanoma and renal cell carcinoma Given the pharmacokinetic success of PEGylated conjugates in terms of improving stability and increasing plasma half- life, it is not surprising that more PEGylated formulations of chemotherapeutics are being advanced into clinical trials. 11

25 12 Unlike polymer- conjugates where the design occurs via chemical modification of parent drug structure, polymeric encapsulation of anti- cancer agents is another design strategy where the structural integrity of the therapeutic agent remains intact. By encapsulation the drug with a biodegradable polymer for instance, one can attain sustained- released drug formulations. Synthetic biodegradable polymers such as poly(lactic acid) (PLA) and poly(lactic co- glycolic acid) (PLGA) have been employed as nano- carriers for sustained released applications 88, 89. The combination of varying ratios of lactic versus glycolic acid influences the degradation kinetics of the polymer and hence release of the parent drug. Naturally occurring polymers such as chitosan have been explored as well 90. Given the popularity of polymeric nano- carriers, there are several carriers have been developed both in pre- clinical and clinical trials 26, 79, 91. Lipid- based carriers include liposomes and micelles. As expected they are biocompatible and biodegradable which are attractive attributes for delivery applications. Like other nano- carriers their sizes and surface functionality can easily be engineered. Doxorubicin was one of the earliest prototypical chemotherapy agents with liposomal formulation 67. Liposomal formulations are currently employed in oncology for delivery of various chemotherapy agents. Today, liposomes are approved by regulatory agencies to carry a range of chemotherapeutics Dendrimers represent a distinct form of polymeric nanomaterials that are designed in the tree- like configuration with repeated branched polymeric units 92. Dendrimers are usually within the 5 nm size range 93. They share similar attributes of other nanomaterials such as biocompatibility and ease of functionalization. Furthermore, dendrimers demonstrate very high solubility and rapid renal clearance 93. The key polymeric building 12

26 13 block polymer that is widely favored for biomedical applications is polyamidoamine(pamam) 94. Dendrimers have been evaluated within the in vivo platform for delivery of chemotherapy agents. For instance successful conjugation and targeted delivery of methotrexate has been established against solid tumors 95. The current status of dendrimer- based carriers remains within the preclinical arena. Carbon- based nanoparticles came into the spotlight with the discovery of fullerenes in Fullerenes represent an allotropic form of carbon composed of 60 carbon atoms within a polygonal configuration. Functionalized fullerenes have been investigated for potential nanomedicine applications. Some of these applications include drug delivery, reactive oxygen species quenching, and MRI contrast agents 96. Carbon nanotubes consists of thin carbon filaments with unique structural and electronic properties that make them ideal as sensor devices for biomedical applications 97. Functionalized carbon nanotubes have also been employed for drug delivery applications especially for anti- cancer drugs as well 97. Inorganic NP such as nanoshells and nanocages represent a distinct class of inorganic NP with practicality in hyperthermia- based therapeutics since they generate heat in response to near- infrared (NIR) light exposure. Nanoshells are usually designed in the nm range and are composed of a silica core surrounded by an outer layer consisting of a metal such as gold. The feasibility of employing nanoshells as imaging, targeting, and therapeutic devices for hyperthermia- based therapy is established 98, 99. Nanocages on the other hand, are smaller compared to nanoshells and engineered from gold. Nanocages are attractive for optimal imaging applications and hyperthermia- based therapeutics given their ease of synthesis and conjugation 100. An obvious shortcoming of inorganic NP when 13

27 14 compared to some of the other polymeric NP carriers is the lack of biodegradability. Hence inorganic NP can accumulate over time in organs thereby raising potential toxicity concerns. Inorganic NP are therefore suited for focal delivery applications. 1.3 GOLD NANOPARTICLES AS PROTOTYPE ANTI- CANCER DELIVERY SYSTEMS OVERVIEW AND SYNTHESIS Gold nanoparticles (AuNP) have recently emerged as attractive tools in nanomedicine in light of their unique optical, biological and surface chemistry functionalization capabilities. Like other metallic NP, AuNP exhibit very strong surface- plasmon- enhanced absorption and scattering effects that are size- dependent in nature 101. Given their strong optic features, AuNP are attractive for imaging and molecular probe applications. The absorption and scatterings effects span both the visible and near infrared (NIR) spectrum range. NIR activity can be exploited for hyperthermia therapy. AuNP also have favorable biocompatibility attributes such as non- toxicity and inertness AuNP also have favorable surface chemistry attributes that facilitate conjugation of biomolecules and other chemical moieties of interest for targeting applications. As a consequence, AuNPs have been successfully employed in cancer targeting 105, 106, colorimetric biosensors , imaging , delivery of therapeutics 113, gene targeting 114, as well as thermal ablation of tumors 98, Given the diverse applications of AuNP in detecting and targeting systemic cancers, AuNP might serve as ideal prototype particulate systems for cancer targeting within the central nervous system (CNS) where targeted therapies are most highly 14

28 15 desirable in light of toxicity vulnerabilities of neural tissues. AuNP can be designed into various configurations of sizes and shapes. Like other nanomaterials, size and shape do play a significant role in both the physical and biological interactions of the nanomaterial. For instance, size and shapes can be optimized such that a large surface area to volume ratio is attained for delivery applications wherein a large payload density is needed. The most commonly engineered particulate shapes entail nanospheres or colloidal gold 121 and nanorods 122. Synthetic strategies for nanomaterials can be classified as either top- down or bottom- up 123. With top- down strategies, nanomaterials are designed on predetermined structural templates based on the proposed the final structure of the material. Hence since predetermined templates are employed, the batch- batch consistency is enhanced. For example, nanorods and nanospheres have been successfully engineered using the template method 122. However, a major limitation with this approach is the difficulty with adapting to high large- scale production of nanomaterials. On the contrary, bottom- up strategies employ seeds as nucleation foci upon which the final NP design is attained and as such are ideal for large- scale synthesis. However, given the fact the ultimate configuration of the nanomaterial is dependent on synthetic conditions in bottom- up strategies, the batch- batch variations may be a limitation. Gold nanospheres are typically designed in the diameter range of <100 nm with absorbance peaks between 520 nm and 550 nm 101. Gold nanospheres are engineered through controlled reduction of gold chloride (HAuCl4) solution as previously described with a few variations 121, 124. The reduction is often accomplished with reducing agents such as citrate that generates nucleation foci for assembly of NP. Other reducing agents 15

29 16 such as sodium borohydride (NaBH4) have been employed with success as well 125. The ultimate diameter of the NP is a function of the extent of reduction. Hence the gold to citrate ratio is a controllable variable that can be exploited to generate a wide range of NP sizes. Gold nanorods can be synthesized through several synthetic techniques. The most commonly employed and favored synthetic technique is seed- mediated synthesis 126, 127. This technique entails generation of gold seeds that will ultimately serve as nucleation foci for designing nanorods. Seeds are often generated using a very strong reducing agent such as sodium borohydride while nucleation requires a weaker oxidizing agent such as hexadecyltrimethylammonium bromide. By controlling the relative amounts of nucleation seeds, one can control the aspects ratio of gold nanorods generated. Another gold nanorod design technique involves electrochemical deposition of gold onto a nano- pore template consisting of polycarbonate or alumina membranes 128. By controlling both the pore diameter and amount of gold deposited, one can control the aspects ratio of gold nanorods. Surface modification of AuNP is often required for clinical applications. Synthesized colloidal AuNP are often capped with citrate as a stabilizer. AuNP are attractive NP systems from a design standpoint since AuNP are highly conducive to surface modifications with biomolecules and polymers. Biomolecular entities such proteins 129 and nucleic acids 130, 131 can be readily conjugated onto colloidal gold by replacing the citrate stabilizing surface cap. In terms of polymeric surface modifications, polyethylene glycol (PEG) appears to be one of the most favored polymers for biomedical applications of colloidal gold especially with respect to drug delivery

30 17 PEG functionalized AuNP are attractive drug delivery systems in light of the favorable biopolymeric attributes of PEG. PEG improves aqueous solubility and stability of AuNP. PEG coating confers stealth properties onto AuNP by preventing rapid- clearance by the reticular- endothelial system (RES) 132, 133. PEG functionality has also been shown to minimize protein adsorption onto the surface of AuNP thereby preventing nonspecific interactions 132, 133. Given that PEG is biodegradable, safe and already FDA approved, it is undoubtedly a critical polymer of choice for AuNP applications within the clinical arena. Moreover, there are several PEG formulated anti- cancer AuNP targeting agents currently in development for clinical trials APPLICATIONS OF GOLD NANOPARTICLES IN ONCOLOGY With properties such as tunable core size, tunable surface chemistry and biocompatible profile, AuNP have emerged as versatile diagnostic and therapeutic tools within the nanomedicine armamentarium 105, 135. Potential oncology applications include diagnostic assays, imaging, hyperthermia cancer therapy, and drug delivery. Given the significant mortality and morbidity of cancer, optimized early detection through the assessment of cancer biomarkers could potentially improve to outcomes. One area of interest has been the development of AuNP- oligonucleotide conjugates as probes for detecting cancer- related protein genes. Such probes have been successfully employed through a variety of techniques including Raman spectroscopy 136, atomic force microscopy 137, 138, and gel electrophoresis for detection of single- nucleotide polymorphisms 139. AuNP can also serve as probes for immuno- assay protein detection Techniques for early detection of cancer cells using colorimetry 108 and surface plasmon resonance of AuNP 143 have been successfully reported as well. Further refinement of AuNP 17

31 18 based detection techniques could have a major impact on early cancer detection and treatment. AuNP have unique optical properties, which make them ideal for in vivo imaging applications for cancer. The surface enhanced Rahman scattering (SERS) properties of AuNP render AuNP as promising contrast agents for spectroscopic imaging 144. The technique is very conducive to multiplexing 145, 146. With SERS, molecules adsorbed onto the surface gold exhibit a marked enhancement of their Rahman scattering. Hence SERS is exquisitely sensitive for detection of single molecules 144, and remains the most promising AuNP imaging application in oncology. Rahman reporters are traditionally fluorescent dyes and they adsorbed onto a gold core. In particular, surface molecules such as CY3 are often employed in light of their favorable Rahman characteristics. In a recent application, SERS conjugated to tumor- targeting ligands successfully targeted tumor markers such as the epidermal growth factor (EGFR) in xenografts tumor models 147. AuNP SERS have been widely employed in studies designed to detect cancer genes 148, 149, cancer protein biomarkers , and intracellular pharmacological targets of anti- cancer drugs 154, 155. The tunable ability of AuNP to absorb light in the near infrared (NIR) range makes AuNP very attractive candidates for hyperthermia therapy against cancer cells. Hyperthermia results in direct tumor cell death and enhanced radiosensitivity 156. Nanoshells are optically tunable nanoparticles composed of a silica core with an ultrathin gold layer 157. They absorb light within the NIR range. In light of their large size >100 nm, nanoshells can selectively accumulate in tumors while sparing normal tissues. Gold nanoshells have been successfully employed in multiple cancer hyperthermia applications 98, 120, These applications include successful elimination of solid tumors in several 18

32 19 animal models 98, 120, 157, 158, and enhance radiosensitivity in human colorectal cancer animal model 159. Hyperthermia destruction of tumors within a canine brain tumor model has also been demonstrated 119. One area of applicable practicality of hyperthermia therapy in humans is the treatment of superficial cancers, notably squamous cell cancer of the head and neck 160. AuNP- directed hyperthermia therapy appears to be successful in head and neck cancer xenograft models 160. Given the initial success of AuNP hyperthermia therapy in preclinical models, there will undoubtedly be an impetus towards clinical translation. A major cancer application of AuNP with significant ramifications is within the area of drug delivery. The favorable attributes of AuNP drug carriers have been exploited for the delivery of peptides , proteins and nucleic acids for gene therapy applications 167, 168. The successful delivery of nucleic acids for targeting aberrant oncogenic pathways creates a novel avenue for treating cancers at the molecular level. AuNPs have been successfully employed for delivery of anti- cancer cytokines such as tumor necrosis factor alpha (TNF- alpha) 116. This is a major advantage given that there are limitations to direct intravenous cytokine therapy secondary to potential severe systemic toxicity. TNF- alpha is a potent cancer- targeting cytokine with similar limitations 169. However, conjugation of TNF- alpha onto AuNP was shown to significantly enhance tumor targeting while minimizing adverse systemic effects 116. AuNP delivery platforms allow for safe and targeted delivery of small molecule chemotherapeutics, which would otherwise have resulted in systemic adverse effects when chemotherapeutics were given intravenously. Methotrexate 170, oxaliplatin 106, paclitaxel 171, doxorubicin 172, and other hydrophobic anti- cancer drugs 173, are examples of chemotherapy agents that have been delivered using AuNP delivery platforms. Given the versatility and broad applications of AuNP drug 19

33 20 carrier platforms, there are currently several AuNP anti- cancer based therapeutics currently being developed for clinical applications 174. AuNP delivery platforms are poised to play a major role in oncology both from a diagnostic and therapeutic standpoint. Their applications for systemic cancers have been well studied. However, extrapolation of AuNP delivery strategies for cancer targeting applications across the blood- brain barrier (BBB) remains a major challenge due to the restrictive nature of the BBB. Further refinements in AuNP design as well as safe BBB disruption strategies may enhance utility of AuNP based therapeutic approaches for cancers within the CNS BLOOD BRAIN BARRIER (BBB) CHALLENGES FOR GOLD NANOPARTICLE DELIVERY PLATFORMS The BBB exerts a significant impediment to targeted AuNP applications within the CNS 7. The endothelial cells of brain capillaries are uniquely interconnected by intercellular protein bridges, called tight junctions, which block the free diffusion of small molecules from the circulation into the brain parenchyma 11. Anatomically, the BBB consists of a contiguous layer of endothelial cells interconnected by tight junctions which are over 100 times more restrictive than capillary systems elsewhere in the body 175. Therapeutics are traditionally transported across the BBB either through passive or active transport mechanisms 176. Passive transport mechanisms favor small (<400 Da), non- polar lipophilic agents 177. On the contrary, polar or water- based compounds are usually transported via active transport mechanisms 177. Furthermore, expression of drug- efflux transporter proteins at BBB provides additional selectivity. Drug efflux proteins can exclude 20

34 21 therapeutics from the brain even if such therapeutics have the appropriate molecule characteristics to cross the BBB. A well- known efflux transporter is the P- glycoprotein, which is a substrate for most chemotherapy agents 178, 179. Hence the design of therapeutic applications destined for the CNS must accommodate and overcome the intrinsic anatomical and physiological constraints of the BBB. The feasibility of designing multifunctional nanoparticles with abilities for BBB transport using techniques such as surface chemistry modification and polymeric encapsulation has been described 26, These strategies all exploit endothelial trans- cellular transport mechanisms. Uptake strategies such as receptor mediated endocytosis, adsorptive- mediated endocytosis and carrier- mediated transport system are potential options for BBB delivery. Examples of receptor mediated BBB therapeutic delivery include; delivery 5- fluorouracyl via transferin receptor 183, and doxorubicin via folic acid receptor 184. BBB transporter systems have also been exploited for nano- based delivery applications. Examples include delivery of α- mannose coated particles via the GLUT1 transporter 185. Lastly, lipophilic surfactants such as polysorbates have been assessed as well for BBB delivery applications In particular, polysorbate- coated doxorubicin nanoparticles were shown to efficiently cross the BBB through adsorptive uptake mechanisms 186, 189. These applications underscore the potential of nanoparticles as chemotherapy delivery agents against malignant gliomas. However, the formidable challenges associated with delivery of AuNP therapeutics across the BBB have been conclusively highlighted in several biodistribution studies When De Jong et al. delivered intravenous AuNP with size ranges between 10 nm to 250 nm into rats, they could only detect gold in the brains of animals treated with the 10 nm 21

35 22 AuNP 190. Moreover only 0.3% of the delivered dose was found within the brain in comparison to 46.3% within the liver 190. Similarly, Sonavane et al. detected gold within the brain of mice treated with either 15 nm or 50 nm AuNPs at very high doses of 1g/kg, but not with 100 nm or 200 nm AuNPs 191. However, the reported amounts represented less than 0.08% of the administered dose 191. Even more intriguing, when polyethylene glycol (PEG) coated AuNPs with sizes of 10 nm and 50 nm were employed by Trentyuk et al. in rats, they did not measure any significant amount of gold in the brain 192. Most recently Lasagna- Reeves et al. examined the biodistribution of daily intra- peritoneal delivery of 12.5 nm AuNPs in mice over 8 days. The AuNP biodistribution within the brain was extremely limited in comparison to the liver or spleen even after serial AuNP administration 193. Hence, based on the above studies, AuNP therapeutics might have limited applications within the CNS unless design features are optimized, or unless the BBB is rendered transport- permissive for AuNP therapeutics. Permeation of AuNP across a transport permissive BBB has not been previously characterized. Therefore, the focus of this dissertation was to examine passive permeation of AuNP across a transport- permissive BBB STRATEGIES TO ENHANCE PASSIVE BBB PERMEATION Since most therapeutic agents do not readily cross the BBB, several strategies to temporally disrupt the BBB have been embarked upon previously (See Table 1.1). Transient disruption of the BBB can be attained using an osmotic agent such as mannitol 194, and alkylated alcohols such as alkyl- glycerol 195. Besides osmotic agents, there are receptor- mediated mechanisms that can enhance permeability of the BBB. The bradykinin receptor analogues such as RMP-7 facilitate delivery of therapeutics into the brain through 22

36 23 enhanced permeability of the BBB 196, 197. Enhanced permeability of the BBB can have both beneficial and deleterious effects. While enhanced delivery of therapeutics is a desired goal, the resulting widespread BBB disruption can have potential deleterious consequences since enhanced permeability could allow toxins into the brain. Hence targeted and focal BBB disruption strategies are highly desirable. If feasible, such strategies could enhance applicability of AuNP based therapeutics given the limited brain biodistribution of systemically administered AuNP. Table 1.1 Enhanced BBB delivery strategies Intra- arterial via carotids Intraventricular delivery Osmotic agents such a mannitol BBB permeation analogues such as bradykinin Convection enhanced interstitial delivery Focused ultrasound (FUS) disruption of the BBB is a novel technique, which results in enhanced permeability of selected regions of interest within the brain, thereby facilitating local delivery of therapeutics The technique entails trans- cranial delivery of low- frequency ultrasound waves, which ultimately result in disruption of BBB 203. Preclinical data suggest FUS disruption of the BBB is safe and often well tolerated FUS has been successfully employed in the delivery of a wide range of therapeutic agents into the brain including therapeutic antibodies 204, chemotherapy 205, and viral vectors

37 24 Given the versatility of FUS with respect to delivery applications, FUS could play a vital role in the delivery of AuNP based therapeutics. However the feasibility of FUS- mediated delivery of AuNP into the brain had not been previously studied and characterized. Therefore one of the objectives of this dissertation was to ascertain if FUS could enhance delivery of AuNP with therapeutic potential across the BBB. 1.4 SCOPE OF DISSERTATION The key objective of this dissertation was to contribute new knowledge about AuNP permeation across the BBB by identifying delivery challenges while simultaneously providing AuNP design based solutions as well as strategies that safely and transiently enhance BBB permeation, hence delivery. It is expected that the findings in this dissertation would enhance future therapeutic applications of AuNP within the CNS. Chapter 2 examines the synthesis and characterization of colloidal gold based on reductive synthetic techniques. We further demonstrate the feasibility of functionalizing AuNP with antibodies, PEG, and nucleic acids (sirna). Chapter 3 addresses permeation of AuNP across transport- permissive BBB as could be seen in malignant brain tumors and other neurological conditions. In particular, this chapter addresses the role of core particle size and PEG functionality in permeation. Small core size in combination with PEG lengths within the MW range appeared to be key permeation determinants. Hence, the potential to optimize AuNP design features in order to enhance AuNP permeation and delivery is realized. 24

38 25 Chapter 4 provides a novel solution for safe and enhanced delivery of AuNP into the CNS even in the most transport- restrictive state of BBB. AuNP biodistribution challenges within the CNS following systemic administration are well established and remain perhaps the most significant hurdle to the successful extrapolation of AuNP therapeutic applications within the CNS. Within the context of the studies done within this chapter, MRgFUS appears as a new paradigm for both enhanced and focal delivery of AuNP. The clinical implications extend beyond tumor targeting and provide new avenues for targeting other neurological conditions. 25

39 26 Chapter 2 2 FEASIBLE SYNTHESIS AND CONJUGATION OF GOLD NANOPARTICLES This chapter describes the synthesis and characterization of colloidal AuNP using citrate, tannic acid and sodium borohydride reductive techniques. The feasibility of conjugating PEG, biomolecules such as small interfering RNA (sirna) and antibodies to AuNPs is discussed. The synthetic techniques employed here formed the basis for AuNP synthesis for subsequent experiments. 2.1 ABSTRACT Amongst nanomaterials, gold nanoparticles (AuNP) are emerging as leading platform candidates for biomedical applications. A major advantage of this nanoparticulate system is the ease of size- tunable synthesis and surface modification. AuNP synthetic techniques have undergone several iterations over the last century. The current reductive synthetic techniques are based on methods previously pioneered by Turkevich and Frens. Using reductive synthetic techniques, we synthesize and characterized AuNP. We also demonstrate feasibility of conjugating polymers and biomolecules onto AuNP. The synthetic techniques described were employed for the design of AuNP for subsequent experiments in Chapter 3. 26

40 INTRODUCTION The biological and physical interactions of nanomaterials are highly dependent particle parameters such as a size and shape 74, 75, , 207, 208. Hence in nanotechnology, the development of robust synthesis techniques that reliably generate nanomaterials of optimized physical dimensions is a paramount goal. Amongst current nanomaterials, gold nanoparticles (AuNP) have emerged as leading nanoplatforms for multiple biomedical applications especially with respect to cancer targeting and treatment 98, 105, 106, , 209, 210. In order to optimize further clinical utility of AuNP, emphasis should be directed towards synthetic endeavors that reproducibly generate gold colloids of consistent sizes and shapes. Synthetic techniques for colloidal AuNP have undergone several iterations within the last 50 years 124, The ideal synthetic approach should allow for predictable synthesis of AuNP of desired sizes and shapes. AuNP can be synthesized using reductive techniques whereby ionic gold is reduced to metallic gold in the presence of a reducing agent. In order to generate homogenously sized particles, a stabilizing agent is often required. The stabilizer can also simultaneously serve as the reducing agent. One of the most commonly utilized AuNP synthetic strategies employs sodium citrate as the reducing and stabilizing agent. This technique was initially described by Turkevich 121. Subsequently Frens demonstrated the feasibility of synthesizing a wide range of AuNP sizes between 10 nm and 150 nm based on controlling stoichiometry of citrate to gold chloride 124. Stabilizers such as thiol have been employed in generating AuNP less than 20 nm 211. Other synthetic strategies have been devised whereby AuNP are generated from precursor seeds which serve as nucleation foci for generation of larger AuNP 213, Seed- based synthetic 27

41 28 techniques can allow for controlled synthesis of AuNP of smaller sizes. In general a stronger reducing agent such as sodium borohydride (NaBH4) 219 or tannic acid 218 is often employed initially in order to generate smaller seeds. Controlled synthesis of AuNP from seeds is then undertaken using a weaker reducing agent, which simultaneously serves as a stabilizer. We were interested in AuNP synthetic techniques based on modifications of the Frens method 124. The technique is simple and the AuNP generated can be easily surface functionalized with polymers such as PEG, small interfering RNA (sirna), and antibodies such as the interleukin- 13 receptor 2- alpha antibody (IL13Ra2). IL13Ra2 is particularly of interest since it is highly expressed in malignant gliomas but not typically expressed in normal brain 220, 221. Hence it could serve as an ideal reagent for targeted gliomas therapies such as tumor- directed cytotoxins 222, 223, targeted viruses 224, tumor vaccine 225, as well as photothermal therapy with silica nanoshell, 118. We therefore assessed the feasibility of conjugating PEG, sirna and IL13Ra2 onto AuNP. The technique of functionalizing AuNP with thiolated sirna has been well described by Giljohann et al 167. Conjugation of IL13RA2 to AuNP is well described in the study by Bernardi et al 118. We employed a variation of the Giljohann technique. Optimization and characterization of AuNP synthesis in this chapter served as a basis for further studies. 2.3 METHODS 28

42 AuNP Synthesis Scheme The AuNP synthesis scheme was previously described by Frens 124. The reducing agents were sodium citrate tribasic dihydrate (Sigma- Aldrich, St Louis, MO), tannic acid(sigma- Aldrich, St Louis, MO) and sodium borohydride (Sigma- Aldrich, St Louis, MO). Sodium citrate and tannic acid was used to generate particles greater than 10 nm, while sodium borohydride (NaBH4) was used to generate sizes less than 10 nm. 1% solutions (W/V) of gold chloride (Sigma- Aldrich, St Louis, MO) and sodium citrate tribasic dihydrate (Sigma- Aldrich, St Louis, MO) were prepared independently by adding 50 mg of solute to 5 ml of H2O in a Falcon tube. For the sodium citrate based synthesis scheme, 1ml of 1% gold chloride (Sigma- Aldrich, St Louis, MO) was added to 90 ml ultra- purified water in an Erlenmeyer flask and stirred with a stir bar over a stir plate (VWR) set to 315 C. The solution was quickly brought to a boil. Immediately upon rapid boiling, 0.4 to 1 ml of a 1% sodium citric solution was added to synthesize the various AuNP core sizes greater than 10 nm. For the tannic acid synthesis scheme, two separate stock solutions were prepared. Stock solution A consisted of 80 ml of distilled water and 1 ml 1% aqueous gold chloride. Solution B consisted of 4 ml of 1% sodium citrate in 16 ml of distilled water with 1 or 2 ml of 1% tannic acid. Both solutions were pre- heated to 60 C and combined together while stirring. The temperature was then increased to 95 C. The reaction was terminated with appreciation of the stable red color of solution. For the NaBH4 synthesis scheme, 0.5 ml of 0.01 M gold chloride was added to 18 ml ultra- purified water, followed by 0.5 ml of 0.01 M sodium citrate at room temperature 29

43 30 while stirring for 5 minutes. In order to generate particles less than 10 nm, 0.4 to 1 ml of 0.1 M sodium borohydride was rapidly added to the above reaction mixture. During the synthesis of AuNP, the color transition from a colorless to a red solution served as the indicator of completion of synthesis. Solutions were allowed to cool at room temperature and filtered with a 0.22 um Millipore filter AuNP Characterization A UV- 1601PC spectrophotometer (Shimadzu UV- 1601PC, Kyoto) with UV Probe software was used to confirm the UV- VIS absorption spectra of our particles. Absorbance measurements were obtained between nm. Hydrodynamic diameter and zeta- potential of particles were then determined by dynamic light scattering (DLS) using a Nano- ZS Zetasizer (Malvern Zetasizer Nano- ZS, Worcestershire, UK) with Dispersion Technology Software (version 5.0). Assessments were made using 500 μl of the samples in plastic disposable cuvettes. PEG- AuNPs were suspended in water and zeta- potential was measured at neutral ph. Transmission Electron Microscopy (TEM) was used for assessment of particle size. Briefly, 10 ul of sample was loaded onto carbon- coated copper grids and images were obtained using Hitachi HD2000 STEM (Hitachi Corp USA, Pleasanton, CA). Particle sizes were measured from TEM using Image J software version 1.39 NIH. Images were initially converted to 8- bit grayscale, and the scale was set appropriately. The imaging thresholds were then adjusted such that only particles were visualized on the image. The particle surface areas were then measured, and their respective diameters were computed from surface area measurements. 30

44 Conjugation of PEG onto AuNP The conjugation thiolated PEG onto AuNP allows for both stability of the AuNP in physiologic media, as well as incorporation of valuable biomolecules and therapeutics. Thiolated moieties are very attractive since they have a natural proclivity for electron sharing with AuNP, hence thiolated groups easily stick onto the surface of AuNP. AuNP were thiolated on the basis of surface area coverage. Surface areas can be ascertained from both the AuNP stock concentration and the extinction coefficient. Concentration can be calculated from maximum absorbance using the Beer- Lambert equation. PEG loading was then based upon surface area (nm 2 ) and the number of PEG molecules per nm 2 of AuNP was computed. Thiolated PEG solutions were prepared as 1 mg/ml working concentration solutions. Using 1 ml of AuNP, various stoichiometric molar ratio of PEG molecule- to- AuNP surface area were conjugated at room temperature. Solutions were stirred and incubated for 12 hours. The resulting PEG- AuNP was suspended centrifuged at 18,000x g in an Avanti Series centrifuge (Beckman- Coulter, Brea, CA). The pellet was washed with ultra- purified water, re- suspended in 1 ml of purified water and filtered using a 0.22 µm syringe filter. Particles were stored at 4 C. In order to assess PEG coverage, electrophoretic mobilities of PEG- AuNP were assessed. Starting with a PEG molecule/nm 2 ratio of 10, we performed 7- serial dilutions. In order to ascertain this, PEG- AuNP and AuNP samples were separated at 100 volts for 30 minutes using 1% (W/V) agarose (Sigma- Aldrich) gel prepared in 0.5X Tris- borate buffer (0.5X TBE) Conjugation of IL13Ra2 Antibody onto AuNP and Uptake in U251 Glioma Cell Lines Conjugation of IL13Ra2 antibody onto AuNP was performed per the protocol described in Bernardi et al 118. The first step entailed conjugation of IL13Ra2 antibody onto 31

45 32 a reactive thiolated PEG called orthopyridyl- disulfide- poly(ethylene glycol)- N- hydroxysuccinimide ester (OPSS- PEG- NHS, 2000 MW, Nektar, San Carlos, CA). The PEGylated antibody is then conjugated onto AuNP in the final step. For the first step, an 81 µmol/l (0.16 mg/ml) solution of OPSS- PEG- NHS was added to a 1 mg/ml solution of IL13Ra2 (clone B- D13, Cell Sciences, Inc., Canton, MA) in volume ratios of 9:1. This solution was allowed to bind overnight at 4 C. Then 15 ul of the PEG- conjugated antibody (13 µg/ml) was added to 1 ml of 2.1 x particles per ml AuNP solution, and incubated for 1 hr prior. Finally 20 µl of 5mg/ml thiolated PEG (SH- PEG MW 5000) was added and the final mixture was incubated overnight at 4 C. A control reaction was also performed whereby AuNP without antibodies was conjugated to SH- PEG. The conjugates were then ultracentrifuged to remove unbound antibody and PEG. In order to assess uptake of conjugates in U251 glioma cell lines, we used gold reflectance microscopy as previously described by Kah et al 226. U251 cells were plated at a density of 5000 cells per well. Wells were incubated for 24 hr at 37 C under separate conditions with bare AuNP, PEG- AuNP, or IL13Ra2- AuNP. Following incubation, cells were trysinized and fixed in 4% paraformaldehyde, and placed on a microscope slide for imaging. Imaging was performed using a confocal microscope (Carl Zeiss LSM510 Meta) in the reflectance mode Conjugation of sirna onto AuNP SiRNA conjugation was based on modifications of previously described protocols for generating AuNP- thiolated sirna conjugates 167. The proposed stoichiometry was 1000 nm sirna per 10 nm AuNP. Therefore 3 ml of a 3.3 nm AuNP (13 nm) solution was spun down and reconstituted to 1 ml for a final AuNP concentration of 10 nm. Similarly 31.5 nmol 32

46 33 pellet of preformed thiolated sirna was dissolved in 315ul for a concentration of 100uM. Thiolated RNA duplexes (1000 nm) were incubated with RNase- free solution of AuNP (10 nm). Following 24 hrs of incubation, 45ul of IL13Ra2- PEG(13 µg/ml) was added to the duplex reaction. An hour later, 60ul of SH- PEG- 5000(5 µg/ml) was added and the resulting solution was incubated overnight at 4 C. The resulting particles were purified by centrifugation at 14,000 rpm for 20 minutes at 4 C. Pellets were washed with RNAse- free water, re- suspended in 1 ml of PBS and filtered using a 0.22 µm syringe filter. Particles were stored at 4 C. Two controls were also synthesized. In the first 10 nm AuNP was conjugated to thiolated 1000 nm sirna without PEG or IL13Ra2- PEG. The second control entailed conjugation of 10 nm AuNP to both thiolated 1000 nm sirna and PEG but not to IL13Ra2- PEG. All conjugated products were characterized via electrophoretic mobility on 1% (w/v) agarose gel as described previously 227. Wells were loaded with 20 pmol equivalents of sirna. The measurement was carried out for 18 min at 100 mv in TAE buffer (40 mm Tris/HCl, 1% (v/v) acetic acid, 1 mm EDTA), and the band with safe fluorescent dye Intracellular delivery of sirna-aunp Conjugates in GFPexpressing U287 glioma cell lines Intracellular delivery of sirna- AuNP conjugates was assessed using confocal fluoresecent microscopy and TEM. Since GFP was expressed by the U251 glioma, we used preformed thiolated GFP sirna with Target Sequence 5'- GGC TAC GTC CAG GAG CGC ACC - 3' (Dharmacon, thermo Scientific, Waltham, MA). In addition, the sirna was tagged with CY3 red for assessment of intracellular delivery. GFP- expressing U287 glioma cells were grown in Dulbecco's modified Eagle's medium (DMEM), with 10% heat inactivated fetal bovine serum (FBS; HyClone Laboratories, Inc, Logan UT) and maintained at 37 C in 5% 33

47 34 CO2. For confocal microscopy, cells were grown to 60% confluency on large coverslips in 6 well plates with 2 ml of media for 24 hr. The media was then discarded and 1800 µl of fresh media was introduced into each well. Cells were treated by introduction of the following conditions: control (200 µl media), sirna alone (200 µl of 1000 nm sirna), AuNP alone(200 µl of 10 nm AuNP), sirna- AuNP(200 µl of 1000 nm sirna conjugated to gold), sirna- AuNP- PEG (200 µl of 1000 nm sirna conjugated to gold and PEG), and sirna- AuNP- IL13Ra2- PEG (200 µl of 1000 nm sirna conjugated to gold, IL13Ra2 antibody and PEG). Following 48 hr of incubation at 37 C in 5% CO2, cells were washed and fixed with 4% formaldehyde and the slides were mounted and observed with a Zeiss Axiovert 200M Spinning Disk confocal microscope (Carl Zeiss Canada, Toronto) equipped with a Hamamatsu back- Thinned EM- CCD camera (Hamamatsu Corporation, Bridgewater, NJ). For TEM, cells were seeded treated as above except no coverslips were required. Cells were trypsizined, centrifuged, and washed with PBS. The supernatant was removed. The cell pellets were fixed in PBS solution (0.1M) containing 2.5% glutaraldehyde for 4h. The cells were dehydrated through an ethanol series and embedded in Epon resin. Thin sections (70 nm) containing the cells were placed on grids and imaged under an 80 kv HD 7000 transmission electron microscope. 34

48 RESULTS AuNP Synthesis The synthesis of AuNP as described by Frens 124 is straight- forward with rapid generation of colloidal AuNP following reduction of ionic gold. As expected the NaBH4 synthesis led to the most rapid generation of AuNP in comparison to citrate. The key variable synthetic parameter was amount of citrate or NaBH4 employed. With tannic acid, the particle size was stable for the amounts employed. However, by varying the amounts of citrate, one could easily modulate the sizes of colloidal AuNP generated (Figure 2.1). We observed generation of smaller sized AuNP with higher concentrations of the reducing agent. Similarly, larger sized AuNP resulted from employing smaller concentrations of citrate. The progress of the synthetic reactions was monitored as evident by the colorimetric changes of the reaction solutions. With the citrate, tannic acid or NaBH4 reduction of gold chloride, complete and successful synthesis of AuNP was characterized by a color transition from a clear solution to one with the color consistency of red wine. Unsuccessful synthesis was characterized by solutions that remained purple in color with persistent appreciable aggregates of AuNP. The aggregates appeared as dark particulate material. Further characterization by UV- VIS spectroscopy and DLS was confirmatory of aggregation in instances where synthesis was unsuccessful. 35

49 36 Figure 2.1. Influence of reducing agent strength on AuNP Size. AuNP size was inversely related to concentration as well as the strength of the reducing agent. Stronger agents like NaBH4 generate much smaller particles compared to a relatively weaker reducing agent such as citrate. Similarly higher concentrations of reducing agents generate much smaller AuNP compared to lower concentrations AuNP Characterization AuNP were characterized by UV- VIS spectroscopy, DLS and TEM. Given that multiple synthetic batches were attempted for optimization, it was much prudent to employ UV- VIS spectroscopy and DLS as the primary characterization modalities. TEM was then used on selected characterized synthetic batches. The UV- VIS plasmon absorption spectra demonstrated absorption λ max ranges from nm (Figure 2.2). The NaBH4 synthesized AuNPs demonstrated λ max at the lower end of the absorption range while citrate synthesized AuNP were on the higher end of the range (Table 2.1). The tannic acid derived AuNP had λ max around 520 nm. In general λ max appeared to increase with increasing particle size. DLS was used to characterize the hydrodynamic diameter (HD) of 36

50 37 AuNP. The NaBH4 derived AuNP had HD around 6 nm. This is expected since NaBH4 is very reactive and ideal for generation of seeds. Tannic acid generated AuNP demonstrated diameters of approximately 10 nm while citrate was ideal for AuNP greater than 10 nm. Particle core size was imaged by TEM analysis for selected batches (Table 2.1). TEM confirmed the generation of nanosphere spheroid- like morphology(figure 2.3 A-B). In general TEM core sizes were smaller than the corresponding HD values. Since all AuNP were stabilized with citrate, the zeta potentials were typically very negative. Table 2.1. Absorbance and HD ranges for AuNP designed from various reducing agents. Reducing Agent λ max Range HD Range (nm) NaBH4 508 < 7 Tannic Acid Citrate > 518 > 18 37

51 38 Figure 2.2. UV-VIS Spectra of AuNP. Representative UV- VIS spectra of synthesized and stabilized AuNP. Strong surface plasmon absorbance peak around 520 nm is appreciated. 38

52 39 Figure 2.3. TEM evaluation of AuNP morphology. Representative TEM of small (A) and large (B) AuNP demonstrating spheroid morphology with Frens method for AuNP synthesis. 39

53 Conjugation of PEG onto AuNP The feasibility of PEG conjugation was assessed given the advantages of PEG surface chemistry for biomedical applications. PEG conjugation occurred rapidly at room temperature. When functionalized with PEG, AuNP solutions demonstrated color transitions from red to reddish- brown. The UV- Vis spectra of PEG- AuNP were characteristic with a rightward shift or increase in λ max with increases in PEG length. In addition PEG- AuNPs absorbed at a higher λ max when compared to their corresponding AuNP. For instance, conjugation of AuNP with HD of 6 nm with PEG of varying length, resulted in λ max increases from 508 nm to 517 nm (Table 2.2 & Figure 2.4). PEG functionalized AuNP demonstrated marked decreased in overall NP surface charge when compared to citrate- stabilized AuNP. Another goal of PEG conjugation was to determine the extent of PEG surface coverage (PEG molecule per nm 2 ). We assessed PEG coverage surface by comparing the electrophoretic mobilities of PEG- AuNP of various PEG- to- AuNP stoichiometric ratios. We were interested in the stoichiometric ratio that significantly retarded the mobility of PEG- AuNP, which would otherwise suggest adequate PEG surface coverage. When 20 nm AuNP were assessed for PEG coverage, electrophoretic mobility was significantly retarded when NP had greater than 0.6 to 1 PEG molecules per nm 2 for 5000MW PEG 5000, and 2.5 PEG molecules per nm 2 for PEG 2000, (Figure 2.5). Hence loading such AuNP at densities greater 2.5 PEG molecules per nm 2 should theoretically provide adequate surface coverage. Similar findings of PEG loading densities were noted for AuNP with 10 nm HD. 40

54 41 Table 2.2. Absorbance and HD changes with AuNP PEGylation. PEG Size (MW) λ max HD (nm) Figure 2.4. UV-VIS Spectra of PEG-modified AuNP. Representative UV- VIS spectra of PEG modified AuNP demonstrating progressive shifts in λmax with increases in PEG length. 41

55 42 Figure 2.5. Electrophoretic mobility of AuNP modified with serial dilution of PEG. (A) Representative agarose gel electrophoresis of 20 nm AuNP modified with serial dilutions of a 10 PEG molecules per nm 2 solution of PEG AuNP mobility is significantly retarded between 0.6 to 1 PEG molecules per nm 2 suggestive of adequate surface coverage. (B) Representative agarose gel electrophoresis of 20 nm AuNP modified with serial dilutions of a 20 PEG molecules per nm 2 solution of PEG AuNP mobility is significantly retarded between 2.5 PEG molecules per nm 2 suggestive of adequate surface coverage. 42

56 Conjugation of IL13RA2 Antibody onto AuNP and Uptake in U251 Glioma Cell Lines We assessed the feasibility of tagging AuNP with the glioma- specific IL13R2A antibody. This antibody may serve as a useful tag for active targeting applications in the future. In presence of OPSS- PEG- NHS, IL13R2A antibody conjugates to AuNP. Once the conjugation process was completed, we employed reflectance microscopy as previously described by Kah et al 226 in order to assess for differential intracellular uptake by U251 human glioma cells. We qualitatively assessed gold uptake in cells treated with either AuNP, PEG- AuNP, or IL13Ra2- AuNP. Our reflectance data was corroborative of cell surface- binding and uptake (Figure 2.6). We observed excellent binding and uptake in U251 glioma cells treated with IL13Ra2- AuNP. Cells treated with PEG- AuNP demonstrated a lesser extent of surface binding. Finally treatment of cells with bare AuNP in media resulted in large aggregates of AuNP. 43

57 44 Figure 2.6 Surface binding and uptake of AuNP, PEG-AuNP and IL13Ra2-AuNP in GFPexpressing U251 human glioma cells. The U251 cells are represented by green while gold reflectance is yellow in confocal reflectance microscopy. (A) Incubation of bare AuNP with U251 cells results in very large aggregates. (B) Incubation of IL13Ra2 functionalized AuNP with U251 cells demonstrates significant surface attachment and internalization of AuNP without significant aggregation. (C) Incubation of PEG functionalized AuNP with U251 cells demonstrates minimal surface attachment and internalization of AuNP without significant aggregation. 44

58 Conjugation of sirna onto AuNP We also assessed the feasibility of conjugating sirna onto AuNP. Since the thiolated chemistry was more favorable for AuNP binding, we followed the protocols for generating AuNP- thiolated sirna conjugates as described by Giljohann et al 167. Generated sirna were assessed by agarose gel electrophoretic mobility as described previously by 227. Song et al demonstrated that binding of sirna onto AuNP at adequate stoichiometric ratios significantly resulted in complete arrest of AuNP electrophoretic mobility 227. We observed a similar phenomenon with our sirna- AuNP conjugates (Figure 2.7). The RNA band was evident only in lanes load with bare sirna. Lanes with various sirna- AuNP conjugates or bare AuNP did not demonstrate an RNA mobility band. Figure 2.7 Electrophoretic retardation analysis of sirna binding. The sirna band is absent in all AuNP/siRNA conjugates at full stoichiometry as previously demonstrated

59 Intracellular delivery of sirna-aunp conjugates in green fluorescent protein (GFP) -expressing U251 glioma cell lines We sought to assess intracellular delivery of sirna- AuNP. Since sirna- based therapies occur within the cell, it was prudent to ensure that conjugates are internalized appropriately. TEM was employed for qualitative demonstration of intracellular uptake of conjugates. When cells were treated with bare AuNP or AuNP conjugated to sirna (sirna- AuNP) without PEG, we noted intracellular uptake of AuNP in aggregates within vesicles that also contained a substantial amount of particulate matter as evidenced by the dark coloration(figure 2.8 A & B). When we employed AuNP with IL13RA2 (sirna- AuNP- IL13Ra2) and PEG (sirna- AuNP- PEG) surface modifications, we also noted intracellular uptake of AuNP. We did not observe any aggregates and the transport vesicles content was for the most part clear in coloration there appeared to be a significant qualitative increase in intracellular uptake of AuNP (Figure 2.8 C & D). 46

60 47 Figure 2.8 Representative TEM internalization of various AuNP conjugates in U251 human glioma cells. Cells were treated with either A) Bare AuNP, B) sirna-aunp, C) sirna-aunp-il13ra2, and D) sirna-aunp-peg. (A) Bare AuNP are internalized as aggregates (arrow) within vesicles with dark contents (scale bar 500 nm). (B) sirna- AuNP are internalized as aggregates (arrow) within vesicles with dark contents (scale bar 500 nm). (C) sirna- AuNP- IL13Ra2 are internalized as within vesicles with clear contents and no aggregation (scale bar 500 nm). (D) sirna- AuNP- PEG are internalized as within vesicles with clear contents and no aggregation (scale bar 500 nm). 47

61 48 As a proof- of- concept, we assessed uptake of a GFP sirna- AuNP conjugate in U251 GFP- expressing cells via confocal microscopy. Our sirna- AuNP conjugates were tagged with CY3- red, which allowed for intracellular visualization of conjugates that were internalized. The sirna was design to target GFP expression. Hence its intracellular transport was expected to decrease cytoplasm GFP expression. In addition, we incorporated IL13Ra2 surface modifications. Examination of cells by confocal microscopy demonstrated internalization of sirna- AuNP conjugates as evidenced by cytoplasmic CY3 red fluorescence. Intracellular CY3 red fluorescence was absent in controls. Furthermore cells that exhibited CY3 punctate intracellular fluorescence, also demonstrated a qualitative decrease or absence in GFP signal (Figure 2.9). However, GFP signal was absent as well in some control cells even in the absence of CY3 red fluorescence. Our GFP- expressing U251 cell lines were clearly heterogeneous in terms of GFP expression and fluorescence. Figure 2.9 Internalization of GFP-targeting sirna-aunp conjugates in GFPexpressing U251 human glioma cells. (A) Controls had no CY3- red labeled tags and as expected there is no perinuclear red fluorescence. Cell population is heterogeneous in GFP (green) expression. (B) CY3- labelled sirna- AuNP conjugates (red) are internalized in a perinuclear (blue) configuration. Almost every cell with perinuclear conjugates (red) demonstrate a marked decrease in GFP (green) signal. 48

62 DISCUSSION AuNPs have broad biomedical and potential clinical applications, which make them leading candidates amongst nano- carriers 98, 105, 106, , 209, 210. Application of AuNP in cancer therapeutics has become an avenue of significant interest. By incorporating targeting ligands, diagnostic markers and therapeutics onto a single AuNP, a potent multifunctional nano- entity can be devised and optimized for effective tumor targeting. In order to accomplish the above objectives, it is imperative to establish protocols that guarantee feasibility of synthesizing and conjugation AuNP with molecules of biomedical utility. AuNP synthetic techniques have been extensively studied and refined over the years. The vast majority of techniques are based upon modifications of reductive synthesis of AuNP from ionized gold as previously described by Turkevich 121 and Frens 124. We generated AuNP using a similar protocol with reducing agents such as sodium citrate, NaBH4, and tannic acid. Of the three reducing agents, NaBH4 generated the smallest AuNP size while citrate generated the largest AuNP. Tannic acid generated AuNP that appeared to be of intermediate size when compared to the products of the other two reducing agents. This trend was not surprising and therefore expected since NaBH4 was the strongest reducing agent and citrate was the weakest. Stronger reducing agents have a tendency to generate smaller AuNP. Hence the strength of a reducing can be exploited for AuNP seed synthesis. With seed synthesis, a stronger reducing agent is employed in order to generate precursor seeds which serve as nucleation foci for generation of larger AuNP 213, Furthermore, varying the concentration of the reducing agent can allow generation of 49

63 50 AuNP with a broad range of sizes. For instance, we were able to generate AuNP of varying sizes by varying the sodium citrate concentration. As expected, smaller AuNP sizes were seen at higher citrate concentrations. In general, NaBH4 appeared to be therefore ideal for applications where seed size (<10 nm) particles are warranted. A major shortcoming of NaBH4 vis a vis sodium citrate is the highly unstable and potentially explosive nature of NaBH4 which requires meticulous precautions when employed for synthesis of AuNP. Citrate on the contrary, is very stable, less toxic and a good stabilizer. Characterization of AuNP is often accomplished via UV- VIS spectroscopy, DLS and TEM. AuNP absorption spectra were characteristic with a surface plasmon absorbance range from 508 nm for small particles to 530 nm for larger particles. DLS analysis demonstrated small hydrodynamic diameters (HD) for NaBH4 derived AuNP, intermediate HD for tannic acid derived AuNP, and larger HD for citrate derived AuNP. TEM analysis confirmed a similar trend. As expected, TEM measurements were typically smaller than the measured HD. TEM evaluates core sizes with focus on the electron density of gold. Hence the citrate- stabilizing corona on the AuNP is not evident on TEM imaging and analysis. Since HD measurements take into account the citrate- stabilizing corona as well as the AuNP core, it is not surprising the particle sizes derived from TEM are smaller than HD measurements. Another objective of this chapter was to demonstrate feasible conjugation of a commonly employed surface polymer PEG; a glioma- specific receptor antibody IL13RA2; and sirna directed against green fluorescent protein (GFP). These were proof- of- principle conjugations, which could undergo further refinement and optimization for future targeted applications. PEG conjugation increased the HD of the AuNP as expected 50

64 51 and also increased λ max. By replacing the citrate- stabilizing corona with PEG we noted a change in surface charge from negative to values closer to zero. Also noteworthy was the observation that a loading density of at least 2.5 PEG molecules per nm 2 was sufficient coverage for AuNP sizes. PEG coverage and its effects on AuNP permeation are optimized and discussed in Chapter 3 where PEG coverage was designed to at least 5 PEG molecules per nm 2. Besides PEG, we also examined conjugation of IL13RA2 onto AuNP since this was a glioma- specific receptor antibody of great interest 118, Bernardi et al had previously demonstrated that IL13RA2 could easily be conjugation onto gold 118. Using the microscopy technique of reflectance, we demonstrated enhanced surface binding and uptake in U251 glioma cell lines when AuNP were conjugated to IL13RA2 antibody. This finding was similar to those of Bernardi et al where they employed gold nanoshells and demonstrated enhanced specific binding and target of glioma cells in a thermal ablation application 118. Our incorporation of reflectance microscopy was novel since it had never been used in assessing gold- antibody conjugates in gliomas. Kah and colleagues described the use of reflectance microscopy as an imaging modality for visualizing surface expression of EGFR on cancer cells 226. Hence, EGFR- tagged AuNP were employed as imaging probes, which would otherwise bind to EGFR- expressing cancer cells and illuminate cancer cells via confocal reflectance microscopy. This technique would prove to be extremely useful for future applications where optimization of active surface targeting of cancer cells is highly desired. Incorporation of sirna onto AuNP remains an active area of research with several protocols, the best described of which entails thiolated sirna constructs 167. Favorable 51

65 52 uptake attributes of AuNP can be exploited for intracellular delivery of sirna without the need for cellular transfection. We were mainly interested in assessing the feasibility of sirna conjugation onto AuNP. In order to accomplish this limited objective, we employed thiolated sirna constructs onto which CY3- red was attached for enhanced intracellular visualization by confocal microscopy. Incorporation of IL13RA2 antibody allowed for enhanced intracellular delivery of CY3- labelled sirna as demonstrated via confocal microscopy. When a CY3- labelled GFP- targeting sirna was used to target GFP- expressing U251 cells, there was a decreased GFP signal in cells that incorporated the CY3- labelled sirna. This would suggest a potential interference with gene expression mitigated by our sirna construct. One caveat is that our U251- GFP expressing cell line demonstrated variable GFP expression, which makes quantitative assessment more challenging. Besides, there are additional challenges to sirna delivery using nano- carriers. Reproducible and efficient conjugation can be difficult to attain at times. Furthermore, internalization of AuNP can also vary. The current consensus seems to suggest that further refinements are warranted in order for sirna- nanocarriers to play a significant role in therapeutics. In summary, we have examined and successfully demonstrated AuNP synthesis from reduced ionic gold. We have also demonstrated the feasible conjugation of PEG, antibody and nucleic acid onto AuNP. The data demonstrate feasible constructs of multifunctional AuNP. These experiments were designed as preliminary proof- of- concept studies. Therefore future optimizations studies are required for successful clinical applications of functionalized AuNP. 52

66 SIGNIFICANCE Through this objective, we successfully synthesized and characterized AuNP using established synthetic protocols based on the techniques of Turkevich 121 and Frens 124. Besides feasible synthesis, we also demonstrated feasible conjugation of PEG, IL13RA2 antibody and sirna. The AuNP synthetic and PEG conjugation schemes derived from this chapter paved the way for studies performed in Chapter 3. The proof- of- concept conjugations of IL13RA2 and sirna support feasible design of multifunctional AuNP. 53

67 54 Chapter 3 3 DESIGN AND POTENTIAL APPLICATION OF PEGYLATED GOLD NANOPARTICLES WITH SIZE- DEPENDENT PERMEATION THROUGH BRAIN MICROVASCULATURE: A BBB TRANSPORT- PERMISSIVE MODEL This chapter describes synthesis and characterization of PEG formulated AuNP and potential applicability for delivery across a transport- permissive BBB. Size- dependent permeation was established suggesting a role for design features in enhancing AuNP delivery across the BBB. This represents the first systematic assessment and characterization of the design influence of PEG surface chemistry and particle core size on AuNP permeation across transport- permissive BBB. This work was published in Etame AB, Smith C, Chan W, Rutka J.T. Design and application of pegylated gold nanoparticles with size-dependent permeation through brain microvasculature. Nanomedicine 7: , Reproduced with kind permission from Elsevier (License Number: ). 3.1 ABSTRACT Gold nanoparticles (AuNP) have gained prominence in several targeting applications involving systemic cancers. Their enhanced permeation and retention within 54

68 55 a transport- permissive tumor microvasculature provides a selective advantage for targeting. Malignant brain tumors also exhibit transport- permissive microvasculature secondary to blood brain barrier disruption. Hence AuNP may have potential relevance for brain tumor targeting. However, there are currently no studies that systematically examine brain microvasculature permeation of polyethylene glycol (PEG) functionalized AuNP. Such studies could pave the way for rationale AuNP design for passive targeting of malignant tumors. In this report, we designed and characterized AuNP with varying core particle sizes (4-24 nm) and PEG chain lengths (MW ). Using an in- vitro model designed to mimic the transport- permissive brain microvasculature, we demonstrate size- dependent permeation properties with respect to core particle size and PEG chain length. In general small PEG chain length (MW ) in combination with smallest core size led to optimum permeation in our model system. 3.2 INTRODUCTION Advances in nanotechnology have significant implications in oncology with respect to diagnostic and therapeutic delivery applications. In particular, nanotechnology- based delivery systems provide a novel avenue for targeting malignant brain tumors where the current prognosis is dismal, by circumventing some of the challenges associated with conventional therapy. Within the context of tumor targeting, nanoparticles (NP) must traverse the porous tumor vasculature in order to deliver their payload onto tumor cells. The unique ability of NP and other macromolecules to permeate and accumulate within tumors has been defined as enhanced permeability and retention (EPR) 30, 31. EPR serves as a selective modality for passive targeting of tumors whereby the porous tumor 55

69 56 microvasculature allows for permeation and retention of NP 30, 31. Similar to their systemic counterparts, malignant brain tumors demonstrate alterations of blood- brain barrier (BBB) integrity resulting in a transport- permissive microvasculature Hence NP mediated strategies in systemic cancers may have potential relevance in malignant brain tumor targeting. Ultimately, the design features as well as core composition of the NP will hypothetically have a significant bearing on the NP permeation and effectively targeting within compromised brain tumor microvasculature. Inert and non- immunogenic NPs such as gold nanoparticles (AuNP) 102, 103 have gained prominence in nano- mediated cancer targeting. Several tumor- targeting105, 106, 209, 210 as well as photo- thermal cancer therapy 98, applications have been demonstrated for AuNP as well. Moreover, there are several PEG formulated anti- cancer AuNP targeting agents currently in clinical trial with promising phase I data 134. The successful extrapolation and application of AuNP anti- cancer therapy to the malignant brain tumors will ultimately require an improved understanding of AuNP permeation through the brain microvasculature. Yet there are currently no studies that have systematically assessed the permeation of PEG- AuNP through permissive transport brain microvasculature. Such information could lead to optimization of permeation design parameters necessary for effective delivery of therapeutics to malignant brain tumors with permissive microvasculature. Given that AuNP are readily amenable to size- dependent synthesis and surface polymeric modifications, they serve as an ideal nano- carrier system for studying physical design influences on permeation. Consequently, given that AuNP have generated a lot of interest because of their tumor- targeting potential for systemic cancers, it was deemed important to ascertain the 56

70 57 permeation profile of AuNP within the brain microvasculature for subsequent malignant brain tumor applications. Therefore, the present study focused on the design of AuNP with various PEG chain sizes, and subsequent assessment of their size- dependent permeation through an in- vitro model of a transport- permissive rat brain microvasculature. Moreover, surface polymeric formulations such as PEG confer several advantages including stability, biocompatibility, and multi- functionality Hence, this study was undertaken to characterize permeation of AuNP through an in vitro platform designed to mimic the brain microvasculature. Prior observations in a flank model of a breast tumor suggested a size- dependent permeation profile of polyethylene glycol functionalized AuNP (PEG- AuNP) 234. In that study, an inverse correlation was noted between permeation and retention. The smaller particles had the fastest permeation kinetics and were less likely to be retained within the tumor compared to larger particles. Based on those observations, we hypothesized that a similar size- dependent phenomenon would be observed if brain microvasculature endothelial cells were permissive to PEG- AuNP permeation. More importantly, we sought to evaluate the permeation effects of PEG chain polymeric design. In order to test this hypothesis in vitro, we employed a co- culture system of fully confluent rat brain endothelial cells (RBEC) and rat astrocytes (RA) designed to mimic the blood- brain interface. Given that the assessment of permissive transport was the goal of our study, we employed a range of gold nanoparticles (AuNP) that encompassed various sizes that have previously been reported to traverse the blood- brain interface following in vivo biodistribution 190, 191. We demonstrate size- dependent permeation of PEG- AuNP with the smallest NP exhibiting the greatest permeation. Furthermore, we demonstrate that PEG 57

71 58 surface polymeric chain was a critical permeation determinant for the smaller NPs when compared to larger NPs within our range. 3.3 METHODS Materials & Cell Culture Rat brain endothelial cells (RBEC) [Cell Applications, San Diego, CA] were maintained in RBEC growth medium (Cell Applications, San Diego, CA) supplemented with 1% Penicillin/Streptomycin (Wisent Inc, Quebec, CA) and grown in dishes coated with a type IV collagen attachment factor (Cell Applications, San Diego, CA). Rat astrocyte (RA) cells (ScienCell, Carlsbad, CA) were maintained in supplemented RA growth medium (ScienCell, Carlsbad, CA). Cultures were incubated at 37 o C in an atmosphere with 5% CO AuNP Synthesis AuNP of various sizes were synthesized as previously described by Frens 124. For particles greater than 10 nm, sodium citrate (Sigma- Aldrich, St Louis, MO) was employed, while sodium borohydride (Sigma- Aldrich, St Louis, MO) was used to generate particles with less than 10 nm. The synthetic schemes described in Chapter 2 were employed for generating AuNP for these studies. 58

72 AuNP Characterization A UV- 1601PC spectrophotometer (Shimadzu UV- 1601PC, Kyoto) was used to confirm the UV- VIS absorption spectra of our particles. Hydrodynamic diameter and zeta- potential of particles were then determined by DLS using a Nano- ZS Zetasizer (Malvern Zetasizer Nano- ZS, Worcestershire, UK). PEG- AuNPs were suspended in water and zeta- potential was measured at neutral ph. Transmission Electron Microscopy (TEM) was used for assessment of particle size. Briefly, samples were loaded onto carbon- coated copper grids and images were obtained using Hitachi HD2000 STEM (Hitachi Corp USA, Pleasanton, CA). Particle sizes were measured from TEM using Image J software version 1.39 NIH. Images were initially converted to 8- bit grayscale, and the scale was set appropriately. The imaging thresholds were then adjusted such that only particles were visualized on the image. The particle surface areas were then measured, and their respective diameters were computed from surface area measurements AuNP PEGylation For pegylation, a 1:5 molar ratio of thiolated- PEG (MW 1000, 2000, 5000, 10000) to particle surface area was added to each AuNP synthesis while stirring at room temperature, and incubated 12 hours to generate PEG- AuNP. PEG- AuNP were collected by centrifugation at 18,000x g in an Avanti Series centrifuge (Beckman- Coulter, Brea, CA). The pellet was washed with ultra- purified water, re- suspended in 1 ml of media and filtered using a 0.22 µm syringe filter. Particles were stored at 4 C. 59

73 Construction of an In-vitro Brain Microvasculature Permeation Model In order to assess permeation of particles across endothelial cells, a well- described Transwell system was employed The system is made of an upper and lower chamber separated by a membrane with pore size (0.4 µm). Briefly 1 x 10 5 RBECs were seeded on the upper membrane while 5 x 10 4 RA cells were seeded at the bottom portion of the membrane. Cells were grown until they were fully confluent at which point the system was ready for transport testing (Figure 3.1). Figure 3.1. Experimental scheme with Transwell double chamber co-culture system of rat brain endothelial cells (RBECs) and rat astrocytes (RAs) designed for assessing microvasculature transport of PEG-AuNPs. The co- culture system, which is designed to 60

74 61 mimic the transport- permissive brain microvasculature, consists of an upper and lower chamber separated by a 400- nm microporous membrane. RBECs are seeded on the upper surface of the membrane, and RAs are seeded on the bottom surface of the membrane. When cells are completely confluent for a couple of days, PEG- AuNPs are introduced into the upper endothelial chamber. At fixed time points, the medium of both chambers is quantitatively analyzed for gold content by inductively coupled plasma- atomic emission spectrometry Assessment of PEG-AuNP Permeation For transport assessment, PEG- AuNP at various concentrations (700 to 5100 ppb) were introduced into the upper chamber with media for 24 hours. Serum- supplemented media from both chambers were subsequently analyzed for gold content by Inductively- Coupled Plasma- Atomic Emission Spectroscopy (Perkin Elmer Optima 3000 ICP AES, Waltham, MA). Transport was ascertained from the relative content of gold in both chambers. Kinetic experiments were similarly performed using the smallest and largest particles with concentration ranges of 1900 to 8000 ppb. Chambers were sampled at 3 h, 10 h, and 20h, and analyzed for gold content as described above Statistical Analysis Data was collected in at least triplicates. ANOVA was initially employed to identify differences between groups. Where differences were observed, Tukey s test was performed as a follow- up analysis for determination of statistical significance. Standard error of mean (SEM) values were employed in generating error bars. Statistical 61

75 62 significance was based on a p< RESULTS Synthesis and Characterization of AuNP We synthesized AuNP with hydrodynamic diameter (HD) ranging from 6-34 nm based on the dynamic light scattering (DLS) measurements. AuNP were synthesized by chemically reducing gold chloride 124 with sodium borohydride and citrate, as previously described to produce particles smaller than 10 nm, and bigger than 10 nm respectively. When particles were further characterized by UV- VIS spectrophotometry, we observed an absorption λ max range from nm. There was an increase in λ max with increasing particle size (Figure 3.2). Particle core size was imaged by TEM analysis, which demonstrated spherical and well- dispersed nanoparticle populations (Figure 3.3 A-D). Based on TEM results, we selected the following AuNPs sizes to conduct our studies: 4 nm, 16 nm, 21 nm, and 24 nm (Table 3.1) all of which were within sizes previously reported to permeate brain endothelial cells following systemic administration 190,

76 63 Table 3.1. TEM cores sizes and corresponding hydrodynamic diameter. TEM Core Size (nm) Hydrodynamic Diameter (nm) Figure 3.2. UV-VIS Spectrocopy Characterization of AuNP. Ultraviolet- visible spectra of citrate- stabilized AuNPs of various core sizes with λmax between 508 and 530 nm. 63

77 64 Figure 3.3. TEM Characterization of AuNP. (A-D) Transmission electron micrographs demonstrating spherical AuNPs with core sizes 4 nm, 16 nm, 21 nm, and 24 nm, respectively. The scale bar in A represents 20 nm; scale bars in B- D represent 100 nm. 64

78 Design and Characterization of PEG-AuNP Non- functionalized AuNP have very limited clinical utility in-vivo since they tend to aggregate and demonstrate rapid clearance by the liver and spleen However, the coating of AuNP with PEG surface chemistry circumvents several of these limitations. We therefore employed thiolated- PEG formulations of varying molecular weights (MW 1000, 2000, 5000, 10000) based on optimization protocols from an earlier study 234. UV- Vis spectroscopy characterization of PEG- AuNP demonstrated an increase in λ max, which is characteristic of PEG functionality (Figure 3.4 A-D). Figure 3.4. Absorption Spectra of various polyethylene glycol (PEG) coated AuNPs. AuNP where coated with PEG 1000, 2000, 5000, & A) 4nm AuNP. B) 16nm AuNP. C) 21nm AuNP. D) 24nm AuNP. 65

79 66 Incorporation of PEG functionality resulted in significant changes in the zeta potential from negative values to values closer to zero (Figure 3.5). Figure 3.5 The effects of PEG surface chemistry on zeta potential. PEG surface chemistry decreases the overall negative surface charge of citrate- stabilized AuNPs. Zeta potential values close to zero are demonstrated by double strikes through the x- axis. 66

80 67 A linear correlation was noted between PEG size and HD for particles smaller than 20 nm (Figure 3.6). Figure 3.6 The effects of PEG surface chemistry on hydrodynamic diameter (HD). Representative graph showing a linear relationship between PEG size and HD. Each line represents the effects of PEG size on the HD of a specific non- PEGylated AuNP. 67

81 68 Finally, PEG functionality was also noted to prevent aggregation and hence promote stability in saline as well as in serum- free culture medium. Aggregates of AuNP were observed in media when PEG functionality was absent (Figure 3.7). There were also changes in absorption spectra in the absence of PEG functionality. When incubated in 1% saline, shifts in λ max were noted (Figure 3.7). Figure 3.7. Aggregation of AuNP in physiological solutions in the absence of PEG surface chemistry. Absorption spectra data demonstrating a shift in absorbance when non- PEGylated AuNPs (4 nm) are incubated with 1% (vol/vol) saline (light graph) versus water (dark graph). Photomicrographs at 100 magnification demonstrate visible aggregates (black) of non- PEGylated AuNPs (4 nm) when incubated overnight at 37 C in serum- free medium within an endothelial cellular background. 68

82 69 When PEG surface chemistry was incorporated, we did not observe any aggregates or changes in absorption spectra (Figure 3.8) Figure 3.8. PEG surface chemistry confers stability and prevents aggregation of AuNPs in physiological solutions. Absorption spectra data demonstrating the absence of shift with PEG surface chemistry (4 nm coated with PEG 1000) when incubated in 1% (vol/vol) saline (light graph) versus water (dark graph) overnight at room temperature. Photomicrographs at 100 magnification demonstrate the absence of visible aggregates when AuNPs with PEG surface chemistry (4 nm coated with PEG 1000) are incubated in serum- free medium against endothelial cellular background. 69

83 In-Vitro Brain Microvasculature Permeation of PEG-AuNP Brain microvasculature permeation of PEG- AuNPs was assessed using a previously characterized model Our data confirm a size- dependent transport of AuNP both with respect to PEG- AuNP core size as well as PEG chain length (Figure 3.9). Figure 3.9. Permissive transport of PEG-AuNP across in vitro rat brain microvasculature model. Bar graphs demonstrating a size- dependent transport profile of AuNPs of different PEG sizes as well as core sizes (n = 3, P <.05). Error bars represent SEM. 70

84 71 The mean percent permeation values and associated SEM data are listed (Table 3.2). Similarly, the statistical significance of permeation profiles between various PEG- AuNPs in terms of p- values from the Anova and Tukey analysis are listed in (Table 3.3). Although all PEG- AuNPs sizes were noted to have brain microvasculature permeation, the most favorable transport profile was seen for the 4 nm PEG- AuNP size, which was statistically different in comparison to the other larger particles (p<0.01). Moreover, a slightly favorable permeation profile was observed when low MW PEG (1000, 2000) formulations of the 16 nm PEG- AuNPs were compared to PEG formulations of the 21 nm, and 24 nm PEG- AuNPs (p<0.05). There was no significant difference in permeation profiles between PEG formulations of the 21 nm, and 24 nm PEG- AuNPs. The effect of PEG size on permeation was also appreciated. In general, whereas PEG formulation size appeared to be a more critical permeation determinant for the 4 nm AuNP (p<0.01), there was no significant effect on permeation of the other larger AuNPs. For AuNPs with core size of 4 nm, there were no significant permeation differences between PEG 1000 and PEG 2000 formulations. However both the PEG 1000 and PEG 2000 formulations had superior permeation profiles in comparison to PEG 5000 and PEG formulations for that particle size. 71

85 72 Table 3.2. Permeation data for PEG coated AuNP. AuNP Core Size (nm) PEG Size (MW) Permeation Percent (± SEM) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

86 73 Table 3.3. Statistical significance between PEG-AuNP permeation profiles from ANOVA and Tukey s test. PEG-AuNP 4P1000 4P2000 4P5000 4P P P P P P1000 N.A. 4P2000 N.S. N.A. 4P5000 p<0.01 N.S. N.A. 4P10000 p<0.01 p<0.01 N.S. N.A. 16P1000 p<0.01 p<0.01 p<0.01 N.S. N.A. 16P2000 p<0.01 p<0.01 p<0.05 N.S. N.S. N.A. 16P5000 p<0.01 p<0.01 p<0.01 N.S. N.S. N.S. N.A. 16P10000 p<0.01 p<0.01 p<0.01 p<0.01 N.S. N.S. N.S. N.A. 21P1000 p<0.01 p<0.01 p<0.01 p<0.01 p<0.05 p<0.05 N.S. N.S. 21P2000 p<0.01 p<0.01 p<0.01 p<0.05 N.S. N.S. N.S. N.S. 21P5000 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.05 N.S. 21P10000 p<0.01 p<0.01 p<0.01 p<0.01 N.S. N.S. N.S. N.S. 24P1000 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.05 N.S. 24P2000 p<0.01 p<0.01 p<0.01 p<0.01 p<0.05 p<0.05 N.S. N.S. 24P5000 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.05 N.S. 24P10000 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 p<0.01 N.S. N.A. = Not Applicable; N.S. = Not Significant 73

87 74 Table 3.3 Continues. Statistical significance between PEG-AuNP permeation profiles from ANOVA and Tukey s test. PEG-AuNP 21P P P P P P P P P1000 N.A. 21P2000 N.S. N.A. 21P5000 N.S. N.S. N.A. 21P10000 N.S. N.S. N.S. N.A. 24P1000 N.S. N.S. N.S. N.S. N.A. 24P2000 N.S. N.S. N.S. N.S. N.S. N.A. 24P5000 N.S. N.S. N.S. N.S. N.S. N.S. N.A. 24P10000 N.S. p<0.05 N.S. N.S. N.S. N.S. N.S. N.A. N.A. = Not Applicable; N.S. = Not Significant Finally, since we observed a size- dependent permeation of PEG- AuNP, we were also interested in their respective kinetic profiles. Given that the most significant difference in transport was noted between the smallest particle (4 nm) and the other three particle sizes tested (16 nm, 21 nm, and 24 nm) as a group, we selected the 4 nm and 24 nm particles as surrogates for transport kinetics. Kinetics was assessed at 3 h, 10 h, and 20 h using relative gold contents of the lower and upper Transwell chambers as indices of transport. The resulting kinetic profile was almost linear for the smaller particle in comparison to the 74

88 75 larger particle (Figure 3.10). Moreover, the smaller particle (4 nm) had a steeper slope than larger particles suggesting faster kinetics. This is in concordance with the permeation data. The linear kinetic profile appears to be consistent with a predominantly passive permeation process, although other associated active mechanisms cannot be entirely excluded. Figure Kinetics of PEG-AuNP permeation across in vitro rat brain microvasculature model. Line graph showing a faster and linear kinetic profile for the smallest core particle size 4 nm when compared to the largest core size 24 nm (n = 3, P <.05). Error bars represent SEM. 75

89 DISCUSSION The current prognosis for patients with malignant brain tumors is very dismal. Similar to solid tumors, malignant brain tumors exhibit disorganized neo- vascularization with concomitant disruption of brain microvasculature integrity. The presence of a porous brain microvasculature around the tumor provides an avenue for selective permeation and passive accumulation of NP through a phenomenon termed EPR 30, 31. Given substantial tumor- targeting potential of AuNPs, for systemic cancers, as well the dire need for novel approaches to brain tumor targeting, the work presented here provides new insight to the contribution of the AuNP core and PEG surface functionality to brain microvasculature permeation. AuNPs with various PEG sizes were employed in this study in light of the favorable biocompatibility attributes of PEG Furthermore, the selection of AuNP core sizes was based upon data from previous biodistribution studies, where particles of the specified core sizes had been shown to permeate the blood- brain interface 190, 191. In general, pegylation of AuNPs resulted slight increases in maximum absorption and overall particle stability when compared to non- PEG AuNPs. A linear correlation was noted between increases in NP hydrodynamic diameter with increases in PEG chain length. Given that we employed a linear PEG polymer, we hypothesize that the linear expansion of HD is most likely a function of PEG orientation on the surface of the nanoparticle. Based on this hypothesis, we would accordingly expect a linear orientation of PEG molecules on the surface of AuNPs, which would allow for a linear correlation between increases in PEG length and increases in particle size. 76

90 77 Another noteworthy consequence of PEG functionality was the significant change in the zeta potential from negative values to values closer to zero. This phenomenon was more pronounced for small NPs compared to large NPs suggesting differential PEG surface coverage. Hence it would appear that smaller NPs would be clinically advantageous and desirable in light of their efficient PEG coverage. While it unclear why this was the case, the observed reductions in surface negative charge was likely indicative of the replacement of citrate surface chemistry by PEG since neutral PEG formulations were employed in our studies. In addition, since neutral PEG formulations were employed, our permeation data was most likely governed by AuNP size as opposed to charge. Finally, PEG functionality was also noted to prevent aggregation and hence promote stability in saline as well as in serum- free culture medium. In the absence of PEG functionality, AuNPs demonstrated exhibited an absorbance shift in 1% saline and a tendency to aggregate in media, both of which would be undesirable properties for in-vivo application. The actual permeation experiments were performed in serum- supplemented media, which would have been more reflective of the in- vivo scenario. Our study did not account for the potential effects of serum proteins on AuNP hydrodynamic diameter. However, we would anticipate a lesser effect given the presence of PEG coating. The permeation of PEG- AuNPs within transport- permissive brain microvasculature is of paramount significance for brain tumor- targeting applications. Based on our model, there was a size- dependent permeation with respect to both NP core size as well as PEG chain length. Of all the core AuNP sizes tested, the 4 nm AuNPs had the most favorable transport and kinetic profile suggesting that particles of such dimensions may be valuable for brain tumor targeting applications. Although the remaining three AuNP core sizes (16 77

91 78 nm, 21nm, and 24nm) also demonstrated permeation through brain microvasculature cells, their respective permeation profiles were not substantially different from each other. These data suggest that when rat brain microvasculature cells are permissive to AuNP permeation, particle core- size is a critical determinant. This is consistent with a prior in vivo study by a co- author, whereby size- dependent permeation of PEG- AuNPs was demonstrated in a breast tumor flank model 234. Size- dependent transport appears to be a very consistent feature of NPs behavior within biological systems especially with respect to cellular 74, 75, 207 and organ uptake , 208. Smaller NPs traditionally have higher permeations, which appear to be consistent with our findings here. Furthermore, our study uncovers a novel finding with respect to PEG chain functionality. Our data suggest that the size contribution from PEG chain functionality is more of a critical permeation determinant for AuNPs with core sizes of approximately 4 nm. PEG chain size did not appear to significantly influence transport for AuNPs with core sizes greater than 16 nm. A hypothesis for this observation may relate to the notion of PEG length- to- particle core diameter ratio. With increases in PEG length, one should expect this ratio to be significantly increased in smaller NPs compared to larger NPs. Therefore, within a size- dependent transport paradigm, increases in PEG length should preferentially influence transport in smaller particles as was observed here. Furthermore, the smaller molecular weight PEG formulations (PEG 1000, PEG 2000) were more readily transported than their higher molecular weight counterparts (PEG 5000, PEG 10000). Thus, it would appear that particles within the 4 nm range would be most amenable to PEG chain size modifications for optimized permeation into the brain microvasculature. 78

92 79 Our data clearly do not address the issue of NP retention, which is an equally important aspect of targeted delivery. Such data can only be established in an in vivo setting. As previously demonstrated, permeation and retention appear to have an inverse correlation with respect to NP size 234. Hence, while smaller NPs have higher permeations, their in vivo retention is not surprisingly low. Lastly, our findings with respect to PEG polymeric chain size design could also have implications in the design of PEG- AuNP suitable for targeting within the brain in other scenarios where the microvasculature is permissive to permeation such as in brain infections. In general, small PEG chain length (MW ) in combination with smallest core size of 4 nm led to optimum permeation in our model system. In summary, our study provides insights on the effects of PEG chain size design on PEG- AuNP permeation in the brain microvasculature using an in-vitro platform. Although our assessment was done in-vitro, we hypothesize that the relative permeations observed would be similar in an in-vivo system that is permissive to PEG- AuNP permeation. Furthermore, our data is consistent with the previous in-vivo tumor permeation study outside the central nervous system 234 using a similar nanoparticle platform. Future studies will entail assessment of PEG- AuNP permeation in murine orthotopic human brain tumor xenografts following intravenous as well as intra- arterial deliveries. 3.6 SIGNIFICANCE Through this objective, we successfully designed and characterized AuNP of various core sizes and PEG length. We observed a size- dependent permeation profile of PEG- AuNP through a transport- permissive brain microvasculature model (Figure 3.11). This 79

93 80 represents the first characterization of the influence of design features (core size and PEG) on AuNP permeation across the BBB and could pave the way for future optimized applications of AuNP- based therapeutics into the CNS via a transport- permissive BBB. Figure Model of PEG-AuNP permeation through transport-permissive brain microvasculature. 80

94 81 Chapter 4 4 ENHANCED DELIVERY OF GOLD NANOPARTICLES WITH THERAPEUTIC POTENTIAL INTO THE BRAIN USING MRI-GUIDED FOCUSED ULTRASOUND IN A PRECLINICAL ANIMAL MODEL This chapter presents a novel approach for focal and enhanced delivery of AuNP with therapeutic potentials across the BBB. The technique of FUS disruption of the BBB is successfully applied and demonstrated to significantly enhance AuNP delivery across the BBB, despite bioavailability limitations associated with systemic delivery of AuNP into the brain. The implications are largely enhanced applicability of AuNP based therapeutics with the central nervous system (CNS). This study represents the first to establish a definitive role of FUS in targeted therapeutic approaches that are based on AuNP platforms. This work was published in Etame AB, Diaz RJ, O Reilly MA, Smith CA, Mainprize TG, Hynynen K, Rutka JT. Enhanced delivery of gold nanoparticles with therapeutic potential into the brain using MRI-guided focused ultrasound. Nanomedicine 2012 Feb [Epub ahead of print]. Reproduced with kind permission from Elsevier (License Number: ) 4.1 ABSTRACT The blood brain barrier (BBB) is a major impediment to the delivery of therapeutics into the central nervous system (CNS). Gold nanoparticles (AuNPs) have been successfully employed in multiple potential therapeutic and diagnostic applications outside the CNS. 81

95 82 However, AuNPs have very limited biodistribution within the CNS following intravenous administration. Magnetic resonance imaging guided focused ultrasound (MRgFUS) is a novel technique that can transiently increase BBB permeability allowing delivery of therapeutics into the CNS. MRgFUS has not been previously employed for delivery of AuNPs into the CNS. This work represents the first demonstration of focal enhanced delivery of AuNPs into the CNS using MRgFUS in a rat model both safely and effectively. Histological visualization and analytical quantification of AuNPs within the brain parenchyma suggest BBB transgression. Lastly we demonstrate successful delivery of AuNP to the tumor- brain invasive front of a rat glioma model. These results suggest a role for MRgFUS in the delivery of AuNPs with therapeutic potential into the CNS for targeting neurological diseases. 4.2 INTRODUCTION BACKGROUND Therapeutic nanoparticles (1 100 nm in diameter) have emerged as promising tools in nanomedicine 113. Gold nanoparticles (AuNPs) are bio- inert and nontoxic which are important features of biocompatible nanomaterials AuNPs have been successfully employed in cancer targeting 105, 106, 209, 210, imaging 112, delivery of therapeutics 113, gene targeting 114, as well as thermal ablation of tumors 98, 104, , 242. These novel targeted diagnostic and therapeutic applications could have significant implications within the central nervous system (CNS) in the treatment of neurological disorders where targeted therapies are most highly desirable in light of toxicity vulnerabilities. However, the blood brain barrier (BBB) provides a significant impediment to 82

96 83 targeted AuNP applications within the CNS. The endothelial cells of brain capillaries are uniquely interconnected by intercellular protein bridges, called tight junctions, which block the free diffusion of small molecules from the circulation into the brain parenchyma. In effect, normal brain capillaries may exclude nanoparticle uptake into the brain that is driven by hydrostatic and osmotic gradients, while passive or active transcellular uptake can still occur. AuNP data from several biodistribution studies in animals with intact BBB highlight delivery challenges associated with the BBB When De Jong et al. delivered intravenous AuNP with size ranges between 10 nm to 250 nm into rats, they could only detect gold in the brains of animals treated with the 10 nm AuNP 190. Moreover only 0.3% of the delivered dose was found within the brain in comparison to 46.3% within the liver 190. Similarly, Sonavane et al. detected gold within the brain of mice treated with either 15 nm or 50 nm AuNPs at very high doses of 1g/kg, but not with 100 nm or 200 nm AuNPs 191. However, the reported amounts represented less than 0.08% of the administered dose 191. Even more intriguing, when polyethylene glycol (PEG) coated AuNPs with sizes of 10 nm and 50 nm were employed by Trentyuk et al. in rats, they did not measure any significant amount of gold in the brain 192. Most recently Lasagna- Reeves et al. examined the biodistribution of daily intra- peritoneal delivery of 12.5 nm AuNPs in mice over 8 days. The AuNP biodistribution within the brain was extremely limited in comparison to the liver or spleen even after serial AuNP administration 193. The above studies clearly underscore the size- dependent and limited nature of AuNP delivery into the CNS for which the intact BBB is the explanation. Therefore, strategies that result in transient permeability of the normal BBB could potentially enhance the biodistribution of 83

97 84 therapeutic AuNPs into the brain for the treatment of neurological diseases wherein the BBB is intact. Such strategies should ideally entail safe and reversible transient increases in BBB permeability to AuNP therapeutics while simultaneously limiting the influx of toxins into the CNS. MRI- guided focused ultrasound (MRgFUS) is a novel technique that selectively and focally disrupts the BBB, thereby increasing its permeability to macromolecules into regions of interest within the brain Intravenously injected lipid- shell perfluorocarbon microbubbles assist with BBB disruption. This non- invasive technique also increases BBB permeability in a transient, non- toxic and reversible manner 200, 201. The BBB disruption induced with MRgFUS typically lasts for 4-6 hrs and has been shown to allow transit of macromolecules up to 150 kda into the brain in mouse and rat models 204, 206, 243. Magnetic resonance imaging (MRI) is used to select the region of brain to be targeted by focused ultrasound and also to visualize the extent of BBB disruption. The feasibility of MRgFUS mediated delivery of macromolecules and therapeutics into the brain has been successfully demonstrated in several studies 102, 118, 207, 230, 236, 242, 244. However, the application of MRgFUS to enhance delivery of AuNP- based nano- carriers into the CNS has not been previously described. Such an application could potentially have significant implications especially given the BBB limitations of CNS biodistribution of intravenously administered AuNPs. Hence, given the significant therapeutic and diagnostic potential of AuNPs within the CNS in conjunction with the selective and targeted capabilities of FUS to overcome delivery challenges across the BBB, this study was undertaken in order to ascertain if FUS could enhance the clinical utility of AuNPs for subsequent CNS applications. We 84

98 85 hypothesized that FUS could alter the conventional biodistribution of AuNPs leading to enhanced delivery within the brain. Accordingly, we assessed the delivery of 50 nm polyethyleneglycol coated AuNPs (PEG- AuNPs) across the BBB with FUS in a rat model since this AuNP size was previously shown to have the best intracellular uptake kinetic profile For the first time, we demonstrate significant enhanced delivery of AuNPs into the brain parenchyma using focus ultrasound. We also conclusively address the issue of AuNP localization within the CNS by detecting AuNPs within brain parenchyma using silver enhancement histology. Lastly we show that our MRgFUS scheme can result in delivery into the CNS that offset the traditional miss- match in AuNP biodistribution between the brain and liver. Using a rat brain tumor glioma model, we successfully targeted the tumor- brain invasive front with AuNP using MRgFUS. Taken together, these results suggest a potential role for MRgFUS for future delivery of AuNP- based therapeutics into the CNS FUS DISRUPTION OF BBB This section includes excepts from work published in Etame AB, Diaz RJ, Smith CA, Mainprize TG, Hynynen K, Rutka J.T. Focused ultrasound disruption of the blood brain barrier: a new frontier for therapeutic delivery in molecular neuro-oncology. Neurosurg Focus Jan;32(1):E3. Reproduced with kind permission from the AANS FUS PRINCIPLES A unique advantage of FUS disruption of the BBB over other conventional BBB disruption schemes, is the selective and regional permeability increases that result in enhanced local delivery within the brain (Table 4.1) 198, The technique entails trans- cranial delivery of low- frequency ultrasound waves, which ultimately result in 85

99 86 disruption of BBB (Figure 4.1) 203. Typically, ultrasonic exposure burst at 10- ms with pressure amplitudes less than 1 MPa are conventionally used for durations of s repeated at the frequency of 1 Hz 244. By employing low frequencies, the chances of permanent tissue damage are minimized. The technique can be employed in conjunction with MRI for both targeting purposes as well as documentation of focal BBB disruption, which is manifest by regional contrast extravasations. Incorporation of intravenously administered lipid- encased perfluorocarbon gas microbubbles (diameter 1 5 μm) further lowers the frequency threshold for BBB disruptions thereby allowing for much lower and safer frequencies to be employed The feasibility of microbubble- assisted FUS disruption of the BBB was first successfully demonstrated a decade ago 201. The FUS BBB disruption effects are not as apparent in the absence of microbubbles since acoustic powers are two orders of magnitude lower 201. As the microbubbles traverse capillaries, they can expand and collapse based on the ultrasonic input. It is hypothesized that FUS results in oscillation and concentration of microbubbles by the capillary walls, which in turn imparts mechanical forces that could result in BBB opening 198, 201, 203. Furthermore, the microbubbles emit acoustic signals that have been highly correlated with BBB disruption in the absence of vascular damage, thus suggesting that acoustic signals could serve as a surrogate for safety 206. The safety of FUS disruption of the BBB is well documented and the overall effects are transient and reversible with no overt neuronal injury The ensuing BBB disruption lasts at most for approximately 4 hours

100 87 Figure 4.1. Schematic of enhanced BBB delivery following FUS disruption of the BBB. Focused ultrasound delivers low- frequency ultrasound waves that cause mechanical oscillations in microbubbles, resulting in disruption of the tight junctions of endothelial cells (ECs) and in enhanced BBB permeability to agents. Table 4.1. Advantages of FUS-mediated therapeutic delivery Focal and targeted delivery minimizes problems seen with widespread BBB disruption Transient disruption of BBB, hence reversible Noninvasive trans- cranial technique Enhanced delivery of chemotherapy, gene therapy, nanomaterials, monoclonal antibodies across BBB 87

101 FUS DESIGN The overall schematic for preclinical FUS systems for BBB disruption is illustrated in Figure 4.2. The animal is anesthetized and positioned supine with the scalp submerged in a chamber containing degassed water. Low frequency ultrasound waves emitted from a focused transducer are transmitted through the degassed water into the cranium. Prior to BBB disruption, animals receive lipid micro- bubbles as well as the therapeutic agent of interest. The targeted area for sonication is selected, followed by burst low frequency ultrasound for BBB disruption. Magnetic resonance imaging (MRI) of the animal brain is obtained prior to and after focused ultrasound BBB disruption. The region of BBB disruption is confirmed on T- 1 contrast- enhanced MRI. 88

102 89 Figure 4.2. Preclinical FUS BBB disruption system. The animal is positioned with the skull partially submerged in a degassed water tank, and microbubbles are intravenously administered. A focused transducer is attached to a network power and personal computer (PC) system delivers low- frequency ultrasound, which disrupts the BBB. MR imaging is incorporated into the procedure both for targeting and confirming BBB disruption. 4.3 METHODS Characterization of AuNP Size- certified 50 nm AuNPs coated with thiolated PEG (MW 2000) were purchased from Nanocs, New York. AuNPs were further characterized with TEM for core diameter assessment and also with agarose gel electrophoresis to document PEG coverage. For the TEM assessment, samples were loaded onto carbon- coated copper grids and images were obtained using Hitachi HD2000 STEM (Hitachi Corp, Schaumburg, Illinois USA). Particle sizes were measured from TEM using Image J software. For the agarose gel characterization, the differential electrophoretic mobilities of PEG coated and non- PEG coated AuNPs were assessed. Samples were run on a 1% agarose gel at 120 mv for 30 minutes and the gel was subsequently photographed MRgFUS Delivery Scheme This study was conducted with the approval of the Sunnybrook Hospital Research Institute Animal Care Committee (Animal Use Protocol #10-281) and in compliance with the guidelines established by the Canadian Council on Animal Care and the Animals for Research Act of Ontario, Canada. Wistar rats weighing g (Charles River, Quebec, Canada), were anaesthetized with inhaled isofluorane for induction. Hair over the dorsal 89

103 90 aspect of the skull was shaved and dilapidated. An angio- catheter was inserted into the tail vein. Maintenance anaesthesia was then achieved with ketamine (40-50 mg/kg) and xylazine (10 mg/kg) and the animal was removed from isofluorane exposure for 5 minutes prior to the start of the experiment. The animal was placed in a supine position with the exposed scalp immersed in degassed water and with its limbs secured (Figure 4.2). The degassed water serves as a conduction media for ultrasound waves emitted from a spherically focused transducer constructed in- house (558 khz, FN=0.8, 10 cm aperture) 245. The transducer is mounted on a MRI- compatible 3D positioning system similar in principle the one described previously 245. Magnetic resonance imaging (MRI) of the animal brain was obtained prior to and after focused ultrasound BBB disruption. The images were obtained using a 1.5 T MRI (Signa 1.5 T, General Electric, Fairfield, CT, USA) set at ETL = 4, FOV = 6 cm x 6 cm, slice thickness = 1 mm, 128 x 128 with T2W FSE parameters being TE = 61.7 ms, TR = 2000 ms, and T1W FSE parameters being TE = 10 ms, TR = 500 ms. Gadodiamide was given at a dose of 0.2 ml/kg prior to and after focused ultrasound BBB disruption to assess the changes in brain vascular permeability. After baseline imaging, animals received 14 mg/kg (by weight of HAuCl 4 ) of 50nm PEG- AuNPs (Nanocs, Inc., New York, NY, U.S.A.), followed immediately by 0.02 ml/kg Perflutren lipid microspheres (Definity, Lantheus Medical Imaging, Inc., N. Billerica, MA, U.S.A) diluted 10:1 in normal saline. The tail vein angio- catheter lines were immediately flushed with saline after nanoparticle and microbubble administration. Focused ultrasound was delivered at the start of microbubble infusion. The target selected for sonication was the parasagittal right frontal lobe. This region was covered using two lines of 4 focal point sonications spaced anterior to posterior in the hemisphere at 2 mm intervals. The two sets of 4 focal point sonications were separated in time by 5 minutes with Definity administration 90

104 91 immediately prior to each sonication set. PEG- AuNP were only administered once prior to the first sonication. The BBB was disrupted using 0.42W acoustic power (approximately 0.26 MPa peak acoustic pressure) in 10 ms bursts at 1 Hz periodic repeat frequency for 2 minutes Experimental Design Animals served as their respective controls for MRgFUS in that the right hemisphere was always sonicated by FUS while the non- treated left hemisphere served as the control. AuNPs were always administered prior to FUS sonication. Animals were observed for 2 hrs and then euthanized with 120mg/kg pentobarbital sodium (Euthanyl, BIMEDA- MTC Animal Health Inc, Cambridge, Canada) via intraperitoneal injection. Blood was collected through cardiac puncture. The liver, spleen, kidney, stomach, and brain were collected. The right and left hemispheres of the brain were separated along the midline interhemispheric fissure, photographed and stored separately. The isolated tissues and blood from animals (n=5) were weighed accurately and were stored at - 20 C until gold content analysis. Another set of Wistar rats weighing 392 to 451 gm were randomly assigned to receive intravenously either 5 mg (by weight of HAuCl 4 ) of AuNPs (n=4) or an equivalent saline volume (n=3) immediately prior to sonication. These animals were allowed to recover from anesthetic and were maintained in a monitored animal facility. During recovery blow- by oxygen at 10 L/min was administered if it was felt the animals showed signs of respiratory distress such as increased work of breathing, excess secretions, or crackles. Animals were observed on a daily basis for signs of neurological impairment including involuntary limb movement, lethargy, weakness, dehydration, and 91

105 92 weight loss. Two doses of Buprenorphine mg/kg (University of McGill) were administered 12 hrs apart during recovery. The animals were allowed to survive for 4 weeks and then sacrificed for assessment of delayed effects of MRgFUS application with and without AuNP administration Histology and Silver-Augmentation for in-situ AuNP Detection Animals underwent light microscopic histological evaluations of the brain, spleen, liver and kidney at 2 h (n=1) and 5 days (n=1) and of the brain at 4 weeks (n=4) after AuNP administration. Tissue preparation and silver- augmentation were carried out by the Pathology Department at the Hospital for Sick Children in Toronto, Canada. Tissues fixed with 3.7% formalin (Sigma- Aldrich, St Louis, Mo) were embedded in paraffin blocks, and then sliced into 5 µm sections. For assessment of delayed pathological changes in the brain at 4 weeks the whole brain was isolated, fixed with 3.7% formalin, and 10 sections spaced 500 µm apart in the mid axial plane on each brain were examined for evidence of necrosis, hemorrhage, vacuolation, neuronal loss, and inflammation. The silver- enhancement protocol from Jackson ImmunoResearch Laboratories, Inc (West Grove, PA, USA) was employed on de- waxed mounted sections of brain, liver, spleen, and kidney. Silver enhancement was quenched after minutes, and sections were then counter- stained with hematoxylin and eosin (H&E). Representative tissue sections were also stained with H&E, but without any silver enhancement as controls. 92

106 Measurement of Brain and Organ Biodistribution of AuNP Gold was extracted from the harvested organs using protocols previously described by Niidome and colleagues 132. Briefly, the harvested organs and blood were digested in aqua regia (3:1 HCL/HNO 3 ) in screw top glass vials. Subsequent evaporation of the aqua regia produced a residue which was dissolved in 3 ml of 0.5N HCl and analyzed by ICP- MS. The ICP- MS analysis was conducted by Maxxam Analytics ( Burnaby, British Columbia, Canada) Statistical Analysis Statistical analysis was performed using PASW Statistics 18 software. Normality of data was confirmed using the Kolmogorov Smirnov test with Lilliefor s correction. A two- tailed, paired t- test (α=0.05, n=5) was used to determine if the difference in gold content between right and left hemispheres was statistically significant. One- way analysis of variance (α=0.05, n=5) was used to determine if the difference in gold content between organs was statistically significant. Homogeneity of variances was assured with the Levene statistics. 4.4 RESULTS Characterization of AuNP Polyethylene glycol (PEG) coating of gold nanoparticles was confirmed with agarose gel electrophoresis. We examined the electrophoretic mobilities of PEG AuNPs and bare AuNPs. In addition to the 50 nm diameter particles, we also employed 10 nm diameter 93

107 94 particles as a control. Polyethylene glycol- coated AuNPs showed no electrophoretic mobility compared to AuNPs stabilized with citrate (Figure 4.3 A). However, the overall differential mobility of 10 nm AuNPs (control) was significantly higher than for the 50 nm particles (Figure 4.3 A). The pegylated AuNPs had a dark brown coloration (Figure 4.3 C). AuNPs were also assessed for core size by transmission electron microscopy (TEM). The TEM demonstrated monodisperse spherical AuNPs (Figure 4. 3 B). Figure 4.3. Characterization of AuNP. (A) Agarose gel (1%) electrophoresis of PEG- and citrate- stabilized AuNPs. PEG- stabilized AuNPs did not demonstrate any mobility on the gel, whereas citrate- stabilized particles migrated. (B) TEM demonstrating spherical AuNPs (scale bar 100 nm). (C) Vial demonstrating the reddish brown coloration of 50- nm PEG- AuNPs. 94

108 MRgFUS Disruption of BBB The schematic for the MRgFUS system is demonstrated in Figure 4.2. For each animal only the right hemisphere was sonicated in the presence of circulating microbubbles. Following micro- bubble assisted disruption of the BBB, MRI gadolinium contrast agent was given and an MRI scan was obtained. Contrast- enhanced T1- weighted (Figure 4.4 A) did not show gadolinium extravasations prior to focal BBB disruption. The corresponding T2- weighted MRI (Figure 4.4 B) prior to BBB disruption was normal. Following MRgFUS, extravasations of gadolinium was demonstrated within the right hemisphere confirming BBB disruption (Figure 4.4 C). Some areas of BBB disruption showed corresponding signal changes on T2- weighted images (Figure 4.4 D). The disruption was carried out within the paramedian aspect of the hemisphere and extended from the convexity to the base of the skull as demonstrated in the contrast enhanced sagittal, and coronal T1- weighted images (Figure 4.4 E-F). A non- homogeneous contrast enhancement pattern within the ultrasound focus was observed in 3 of 5 animals in which orthogonal magnetic resonance imaging was performed. The average spacing between peak contrast intensity in these animals 0.16 cm (n=3) corresponded well with the half wavelength of the ultrasound (approximately 0.13 cm), suggesting standing wave influence. 95

109 96 Figure 4.4. MRI demonstrating disruption of BBB by MRgFUS. (A) Contrast- enhanced axial T1- weighted images prior to MRgFUS do not show extravasations of gadolinium. (B) T2- weighted axial image prior to MRgFUS shows no increased signal. (C) Contrast- enhanced axial T1- weighted images following MRgFUS show extravasations (arrow) of gadolinium in the right hemisphere (R) but not the left hemisphere (L), suggesting BBB 96

110 97 disruption. (D) T2- weighted axial images following MRgFUS show increased T2 signal (arrow) corresponding to area of BBB disruption in the right hemisphere (R). Contrast- enhanced coronal (E) and sagittal (F) T1- weighted images following MRgFUS show extravasations of gadolinium (arrow) in the right hemisphere, confirming BBB disruption Gross Pathological Examination of the Brain The gross pathology of the brain was examined 2 hours after BBB disruption. The sonicated right and non- sonicated left hemispheres were sectioned along the midline interhemispheric fissure. Examination of the medial aspects of the hemispheres clearly demonstrates evidence of punctate extravasations seen as reddish- brown deposits within the sonicated hemisphere whereas the non- sonicated hemisphere has a normal appearance (Figure 4.5 A). The extravasations represent disruption of the BBB in focal areas within the treated hemisphere. This was further confirmed by silver enhancement histology (Figure 4.5 B). 97

111 98 Figure 4.5. Gross pathology and histological demonstration of BBB disruption by MRgFUS. (A) Gross pathology of the brain showing areas of extravasations (arrow) suggestive of BBB disruption within the right paramedian hemispheric surface that underwent MRgFUS. The left paramedian hemispheric surface appears normal this hemisphere was not treated with MRgFUS. (B) Demonstration of AuNP extravasations (arrow) by silver enhancement and H&E histology within the right brain hemisphere (scale bar 50 µm). 98

112 Histological Examination of The Brain The hemispheres were examined by H&E histology as well as silver augmentation techniques. Silver augmentation is a well- established technique for visualizing AuNPs in H&E sections. Coronal and axial sections were examined. A representative coronal section at low magnification shows (arrow) areas of extravasations within the sonicated right hemisphere but not within the non- sonicated left hemisphere (Figure 4.6 A). Silver augmentation in combination with H&E demonstrates AuNPs within the perivascular spaces as well as within the brain parenchyma (arrow) following MRgFUS treatment of the right hemisphere (Figure 4.6 B). AuNPs can be visualized (arrow) approximately 150 µm from the site of disruption (Figure 4.6 B). On the contrary, the AuNPs within the non- sonicated left hemisphere localized mainly within intravascular compartment without any appreciable localization within the brain parenchyma (Figure 4.6 C). 99

113 100 Figure 4.6. CNS localization of AuNP following of BBB disruption by MRIgFUS. (A) H&E histology of coronal section of the right and left frontal lobes. The area (arrow) of BBB disruption by MRIgFUS within the right frontal lobe is shown (scale bar µm). (B) Demonstration (arrows) of peri- vascular and brain parenchyma localization of AuNP by 100

114 101 silver enhancement and H&E histology within the right frontal lobe of CNS following MRIgFUS. AuNP can be seen up to distances of 150 µm from initial site of BBB disruption (scale bar 50 µm). (C) Demonstration (arrow) of intra- vascular localization of AuNP by silver enhancement and H&E histology within the left frontal lobe of CNS in the absence of MRIgFUS. There was evidence of brain parenchyma (CNS) localization in left frontal lobe (scale bar 50 µm). At 4 weeks after focused ultrasound application to the right hemisphere 2 of 7 animals demonstrated small resolving subcortical hemorrhage (Figure 4.7 A). However, no isolated areas of necrosis, inflammation, or neuronal loss were found (Figure 4.7 B). Remnant perivascular and parenchymal AuNPs were present 4 weeks after MRgFUS delivery (Figure 4.7 C). 101

115 102 Figure 4.7. Brain histology in the subacute phase after BBB opening for delivery of AuNPs. (A) Resolving hemorrhage in the subcortical zone of the right frontal lobe (scale bar 2 mm, star marks right hemisphere). (B) Axial section at the level of the temporal lobes demonstrating normal brain structures (scale bar 5 mm, star marks right hemisphere). (C) AuNPs in the periventricular and parenchymal location (arrows) with no associated hemorrhage, necrosis, or inflammation (scale bar 50 μm). 102

116 Histological Examination of The Liver, Spleen, and Kidney Histological sections of liver, spleen, and kidney of treated animals were performed in order to assess for any significant changes. There were no histological abnormalities noted (Figure 4.8 A-C). Using silver augmentation, AuNPs were visualized within the spleen, liver, and kidney. The most significant AuNPs visualization was noted within the spleen, which corresponded to the maximum gold concentration per gram of organ tissue recorded by inductively coupled mass spectroscopy (ICP- MS) amongst all the organs tested. 103

117 104 Figure 4.8. Spleen, liver and kidney histology following AuNP CNS delivery by MRIgFUS. (A) Demonstration of AuNP by silver enhancement and H&E histology within the spleen following MRIgFUS. The AuNP are shown (double arrows) within spleenic macrophages (scale bar 50 µm). (B) Demonstration (arrow) of AuNP by silver enhancement and H&E histology within the liver following MRIgFUS (scale bar 50 µm). Demonstration (arrow) of AuNP by silver enhancement and H&E histology within the kidney following MRIgFUS (scale bar 50 µm). 104

118 Qualitative Assessment of AuNP Content Within The Brain Animals were intravenously given 50 nm PEG- AuNPs (14mg/kg) via the tail vein and the right hemisphere was sonicated with MRgFUS. Two hours after BBB disruption animals were euthanized and each hemisphere was assessed for gold content by ICP- MS. The ICP- MS data show a significant difference between the gold content of the sonicated right hemisphere versus the non- sonicated left hemisphere. MRgFUS treatment of the right hemisphere resulted in over 3- fold enhancement of AuNP delivery when compared to the left hemisphere brain (1593 ± 190 ng Au/gm brain S.E.M. Right Hemispheres versus 474 ± 46 ng Au/gm brain S.E.M. Left Hemispheres, P = 0.007) (Table 4.1 & Figure 4.9). Table 4.1. Enhanced CNS biodistribution of AuNP with 2hr Brain Hemisphere AuNP Amount (ng/gm ± SEM) Right sonicated (FUS) 1593 ± 190 Left - unsonicated 474 ±

119 106 Figure 4.9. Enhanced CNS biodistribution of AuNP 2hr following MRIgFUS. Following tail vein injection of AuNP, the FUS sonicated right hemisphere had a 336% enhanced uptake in comparison to the non- sonicated left hemisphere (n=5, P = 0.007). Error bars are standard errors Biodistribution of AuNP Outside the CNS The biodistribution of 50 nm PEG- AuNP outside the CNS was also assessed at 2 hours after BBB disruption similar to the assessment for the CNS content (brain in particular). The gold content of the spleen, liver, kidney, and blood for each animal were measured by ICP- MS and reported in ng/gm of organ. The highest amount of gold was seen within the spleen (20760 ± 4097 ng/gm, S.E.M), followed by blood (4796 ± 4032 ng/gm S.E.M), kidney (3914 ±1225 ng/gm, S.E.M) and liver (1685 ± 990 ng/gm, S.E.M). Gold content was significantly different between organs (df = 3, F = , P < 0.001) (Table 4.2 & Figure 4.10). 106

120 107 Table 4.2. Biodistribution of AuNP outside the 2hr Organ AuNP Amount (ng/gm ± SEM) Spleen ± 4097 Liver Kidney Blood 1685 ± ± ± 4032 Figure Biodistribution of AuNP outside the CNS 2 hrs following MRgFUS. The largest amount of AuNPs following tail vein injection was noted in the spleen, followed by kidney, blood, and liver. Error bars are SEM (n = 5, P b between group ANOVA). 107

121 DISCUSSION The intact BBB is a major impediment to the delivery of therapeutics into the CNS for the treatment of neurological disorders. Therefore, novel delivery strategies that can overcome the traditional challenges associated with the BBB have the potential to significantly impact the diagnosis and subsequent targeting of disease processes within the CNS. Nano- delivery platforms serve as attractive carriers in light of their small size as well as their unique abilities to traverse biological membranes. In particular, AuNP platforms have been employed in multiple practical therapeutic and diagnostic applications outside the CNS 98, 105, 106, , 209, 210. However, there are significant limitations in CNS bioavailability of AuNPs following intravenous administration based on animal biodistribution studies Therefore, in order to improve applicability of AuNP delivery platforms within the CNS, we employed MRgFUS - a novel technique that has been shown to transiently disrupt the BBB and potentially deliver therapeutics into the brain118, 242, 244, To the best of our knowledge, this study represents the first demonstration of focal enhanced delivery of AuNPs into the cerebral hemisphere. We used MRgFUS parameters that had been previously optimized and well tolerated in small animals 245. The study was designed such that each animal would serve as its own control. The right hemisphere of the brain was sonicated with MRgFUS while the left hemisphere was not, and served as control. By quantifying the amount of gold within both hemispheres of the brain, we were able to show that MRgFUS alters the AuNP biodistribution within the sonicated right hemisphere to comparable levels like the liver within 2 hrs of administration of AuNP. Hence a significant amount of AuNPs can be delivered into the brain with MRgFUS within a 108

122 109 short period of time, which would not have been otherwise possible by conventional means. In addition, MRgFUS appeared to limit the fraction of AuNPs that would have otherwise been sequestered in the spleen and liver. Using silver augmentation techniques, we detected AuNPs within the brain parenchyma of the MRgFUS treated right hemisphere. There were areas of red blood cells (RBCs) extravasations in conjunction with AuNPs, but we also observed gold within the brain parenchyma even in the absence of RBC extravasations in the acute (2 hr) and subacute period (4 weeks). This would suggest that AuNPs could be safely delivered across the BBB without RBC extravasations. Areas of red blood cell extravasations in the region of BBB disruption clustered in white matter tracts, which may be indicative of regional variability in the brain vasculature response to a set peak sonication pressure or effects of non- homogeneity in microbubble size. Furthermore, we were able demonstrated that FUS can deliver AuNPs to distances as far as 150 µm from the transiently disrupted BBB without any associated extravasations of RBCs (Figure 4.6). This is particularly important since therapeutics can be delivered further into the brain thereby overcoming diffusion- related limitations within the brain parenchyma. The extensive migration of AuNPs into the brain parenchyma could be attributed to the combined ballistic effects of bubble oscillation, radiation pressure and acoustic streaming. In addition, enhanced diffusion across the extracellular space following BBB disruption could also play a secondary role. The exact localization of AuNPs within normal brain, parenchyma versus intra- vascular space, has not been defined previously. We have observed AuNPs in the capillaries within normal brain and extravasations of AuNPs into the brain parenchyma in the region of focused BBB disruption. Parenchymal localization of AuNPs has significant therapeutic 109

123 110 relevance because if AuNPs are restricted to the intra- vascular space, then that would of necessity limit their clinical utility for targeted delivery application into the brain parenchyma. We demonstrated both peri- vascular and intra- parenchymal localization of AuNPs with the MRgFUS- treated right hemisphere suggesting that the AuNPs have transgressed the BBB. On the contrary, the non- treated left hemisphere had AuNP localized to the intravascular space and AuNPs were cleared from the intravascular spaces by 4 weeks. Interestingly this non- sonicated hemisphere also had measurable AuNP content by ICP- MS although there was no detectable gold within the brain parenchyma. These findings suggest that the ICP- MS quantified AuNP content from previous biodistribution studies might have as well been reflective of intravascular space localization as opposed to localization within the brain parenchyma as has been previously reported in prior biodistribution studies Lastly, MRgFUS opened the BBB in focal areas as is evident from the AuNP deposition within the brain. Focal opening of the BBB is more advantageous compared to the non- focal widespread BBB disruption that is encountered with osmotic agents employed for a similar purpose. MRgFUS therefore translates into targeted delivery, which in itself is a necessary requirement for advancing AuNP platform therapeutics within the CNS to treat neurological disorders. Given the potential for induction of seizures, hemorrhage, and cerebral edema after BBB disruption we observed a set of rats in which received MRgFUS disruption with the same ultrasound parameters as the group of animals in which gold uptake was quantified. The survival of these animals (n=7) to 4 weeks with no evidence of seizures or neurological deficit shows that focal transient disruption of the BBB with MRgFUS was safe. Delivery of AuNPs into the CNS by MRgFUS in four of these animals did not result in any neurological 110

124 111 deficit or pathologic changes on brain histology secondary to vascular occlusion or induction of inflammatory response. We did however observe resolving hemorrhagic foci in the sonicated hemisphere in some animals. From our previous experience, the intensity of gadolinium enhancement can be a marker for the degree of BBB disruption. Comparing the peak intensity observed in our study to prior studies suggests that we may be able to reduce the sonication peak pressure even further, allowing transient blood brain barrier disruption with lower risk of immediate complication such as hemorrhage. Furthermore, we were also interested in the effects of MRgFUS on the biodistribution profile of the AuNPs. Traditionally, AuNPs concentrate in the liver and spleen from systemic delivery. This effect appears to be less with PEG coated AuNPs. As expected, most of our AuNPs were found in the spleen followed by the blood after 2 hrs. However, the amount of AuNPs within the liver was comparable to the MRgFUS sonicated right hemisphere when normalized for the mass of the organ. This would at least suggest that MRgFUS mediated- delivery significantly minimizes the traditional AuNP biodistribution mismatch that exists between the liver and brain. Furthermore since this was assessed only at 2 hrs post treatment and a substantial amount of AuNP was still present in blood, it is possible that additional AuNPs would have accumulated in sonicated hemisphere brain over time. Moreover, because we normalized our AuNP values based on the entire right hemisphere mass as opposed to the focal volume of BBB disruption within the hemisphere, we may have underestimated the relative amount AuNPs delivered into the region of brain with BBB disruption. Ideally, one would acquire a more accurate representation of the enhanced focal delivery of AuNP by normalizing the gold content to the volume of brain that demonstrates BBB disruption after sonication. However, 111

125 112 volumetric image acquisition in our system was limited due to resolution constraints associated with imaging the small brain of rats. Another potential approach to circumventing the effects of the spleen and liver on AuNP biodistribution would be intra- arterial administration via the carotid artery. A major limitation of conventional delivery of large molecules such as 50 nm AuNPs is compromised diffusion within the extracellular spaces of the brain. The size constraint of normal brain extracellular space has previously been predicted at nm based on in vivo diffusion analysis with quantum dots and dextrans 250. Nonetheless, we employed 50 nm AuNPs since this particle size has been shown to have the best intracellular uptake kinetic profile 74-76, which is advantageous for therapeutic delivery. Through MRgFUS we were able to circumvent diffusion- related limitations by demonstrating focal delivery of 50 nm AuNPs across the blood- brain barrier further into brain parenchyma. Once focally delivered, the favorable uptake kinetics of 50 nm AuNPs can be exploited for intracellular delivery of chemotherapy, small interfering RNAs (sirnas), peptides and other macromolecules of therapeutic value. In addition, given the successful delivery of 50nm AuNPs, one should anticipate marked enhanced delivery of smaller particles with optimal passive diffusion profiles. In summary, our work represents the first demonstration of focal enhanced delivery of AuNPs with therapeutic potential into the cerebral hemisphere using MRgFUS in a rat model. Based on silver enhancement histology, this work also provides the first direct evidence of localization of AuNPs within the brain parenchyma suggesting BBB transgression. Lastly, we also show that MRgFUS could significantly minimize the AuNP biodistribution mismatch that traditionally exists between the liver and brain. Taken 112

126 113 together, these results suggest a potential role for MRgFUS in the delivery of AuNPs with therapeutic potential into the CNS for targeting neurological disorders. 4.6 SIGNIFICANCE The work described in this chapter was the first to establish a definitive role of FUS in delivery of AuNP with therapeutic potential into the brain. This work demonstrates that FUS can significantly ameliorate the limitations associated with CNS biodistribution of systemically administered AuNP. Furthermore, the selective increase in BBB permeability afforded by FUS may be exploited to target invasive malignant brain tumor cells in areas where the BBB appears to be intact. Traditionally, invasive tumor cells may escape anti- cancer chemotherapeutics in areas where the BBB is intact and such cancer cells may form the basis for disease recurrence and poor clinical outcomes. Hence by focal targeting of this invasive front of tumor cells, FUS- mediated delivery of AuNP based anti- cancer therapeutics could have a major impact in the treatment of malignant brain tumors. Given the successful application of FUS in delivery AuNP with therapeutic potential across the BBB, we sought out to ascertain if FUS could target the invasive front of tumor cells in a rat glioma model. The goal was to disrupt the BBB around the margins of the tumor where the BBB was still presumably intact. The rat brain tumor model was established by injecting 160,000 glioma (9L rat gliosarcoma) cells into the right frontal lobe of Wistar rats. A 1.5 Tesla MRI was employed in order to confirm both the presence of tumors and subsequent targeted disruption of the BBB with FUS (Figure 4.11). Tumor- bearing animals were injected with 50 nm AuNP (~ 14 mg/kg) followed by microbubble- assisted FUS disruption of BBB ( khz). Using silver stain histology we demonstrated successful delivery of AuNP around the tumor margins with extension into 113

127 114 the brain parenchyma where the tumor invasive front is situated (Figure 4.12), whereas no delivery was noted in control animals that did not received FUS (Figure 4.13). Figure Magnetic resonance imaging of MRgFUS disruption of the BBB around a brain glioma tumor graft. Coronal Gd- enhanced T1- weighted MR images of rat brain before (left) and after (right) sonication. Contrast enhancement around the sonicated right hemispheric tumor increases after sonication (bright signal). The nonsonicated left hemisphere lacks contrast enhancement. 114

128 115 Figure FUS- mediated delivery of AuNP to the invasive front of a rat glioma tumor graft. AuNP were delivered intravenously after FUS sonication of the peri- tumor region and adjacent brain parenchyma. Using silver stain histology, AuNP can be seen in the peri- tumor region (single arrow) where tumor cells are invading the adjacent brain parenchyma. The primary tumor mass can be appreciated on the left (double arrows). 115

129 116 Figure Perivascular localization in AuNP in rat glioma tumor graft in the absence of FUS. AuNP were delivered intravenously without assistance of FUS disruption of BBB. Using silver- stain histology, AuNP (double arrows) are most seen within the tumor in a perivascular location. No AuNP are seen within the brain parenchyma around the tumor. 116

130 117 Our observations suggest that FUS can provide focal and targeted delivery of AuNP therapeutics to the invasive front of malignant gliomas, and therefore may become a very important delivery paradigm for molecular- based anti- glioma therapeutics in the future (Figure 4.14). Figure MRgFUS model system for preclinical delivery of AuNP into the brain. 117

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