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Photothermal therapy of prostate cancer using gold nanoparticles Leung, Jennifer Ping 2010

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PHOTOTHERMAL THERAPY OF PROSTATE CANCER USING GOLD NANOPARTICLES  by  Jennifer Ping Leung  B.Sc., University of Calgary, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES  (CHEMISTRY)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  November 2010  © Jennifer Ping Leung, 2010  Abstract Prostate cancer is the most common cancer in men. Many existing treatments for prostate cancer are often not completely effective and/or are invasive. Multimodal or combination therapy represents a promising new method to fight disease. Therefore, a combination of different therapeutic strategies may be the best alternative to improve treatment outcomes for prostate cancer. Photothermal therapy combined with gene and chemotherapy was proposed as a novel approach to treatment. In this work, photothermal therapy with gold nanoparticles and near infrared laser (NIR) irradiation, and gene therapy targeting heat shock protein 27 using antisense oligonucleotides (ASO), was investigated. Four types of small (<100nm), NIR absorbing gold nanoparticles (nanoshells, nanorods, core-corona and hollow nanoshells) were synthesized using wet chemical methods and characterized by transmission electron microscopy, dynamic light scattering and UV-vis spectroscopy. Their synthesis and properties were evaluated, to determine their feasibility as a photothermal agent for clinical applications. In vitro cellular uptake studies of the nanoparticles into prostate cancer cell lines, LNCaP and PC3, were measured using light scattering microscopy. The effect of ASO on hyperthermia of cancer cells was investigated using cell viability assays to determine if heat sensitization was possible. Photothermal treatment of the cancer cell lines using synthesized gold nanoparticles was evaluated and compared with commercially available Auroshell® particles. The effect of photothermal treatment and ASO in vitro was determined by measuring cell viability. A preliminary in vivo model study was performed to examine treatment conditions and heat generation. Small gold nanoshells (40nm) had good photothermal properties and the greatest cellular uptake, and were used in photothermal studies. Under 4W laser irradiation, an increase in temperature of 12C and decrease in cell viability of up to 70% were obtained. Comparative work suggests that the 40nm gold nanoshells have a higher therapeutic efficiency than the larger Auroshell® particles. It is uncertain from the present study, if the addition of ASO can sensitize prostate cancer cells to hyperthermia. However, ASO treatment had a concentration dependent effect on cell viability, which could be useful to treatment goals. In vivo, localized high temperatures were generated with gold nanoparticles at low laser powers.  ii  Preface Preliminary in vivo work appearing in Sections 5.2.4. and 5.3.3 was conducted in the UBC LASIR laboratory. The mice were handled by an animal care technician, Virginia Yago (Prostate Center). I helped coordinate the experiments, recorded the temperature data for the photothermal treatment and analyzed the results. Approval was obtained from the UBC Animal Care Committee (Certificate Number: A100165).  iii  Table of Contents Abstract ................................................................................................................................... ii Preface .................................................................................................................................... iii Table of Contents ................................................................................................................... iv List of Tables .......................................................................................................................... vi List of Figures........................................................................................................................ vii Acknowledgements ................................................................................................................. ix Chapter 1. Introduction .......................................................................................................... 1 1.1 Prostate Cancer ................................................................................................................ 1 1.2 Prostate Cancer Treatment Modalities ............................................................................. 2 1.3 Project Purpose................................................................................................................ 3 Chapter 2. Gold Nanoparticle Synthesis and Characterization ............................................ 4 2.1 Introduction ..................................................................................................................... 4 2.2 Materials and Methods .................................................................................................... 6 2.2.1 Materials ................................................................................................................... 6 2.2.2 PEGylation ............................................................................................................... 6 2.2.3 Methods of Purification ............................................................................................ 7 2.2.4 Characterization ........................................................................................................ 7 2.3 Synthesis of Gold Nanoshells .......................................................................................... 9 2.3.1 Introduction .............................................................................................................. 9 2.3.2 Preparation of Gold Nanoshells................................................................................10 2.3.3 Results .....................................................................................................................12 2.4 Synthesis of Gold Nanorods ...........................................................................................17 2.4.1 Introduction .............................................................................................................17 2.4.2 Preparation of Gold Nanorods ..................................................................................18 2.4.3 Results .....................................................................................................................19 2.5 Synthesis of Gold Core-Corona Nanoparticles ................................................................22 2.5.1 Introduction .............................................................................................................22 2.5.2 Preparation of Gold Core-Corona Nanoparticles ......................................................24 2.5.3 Results .....................................................................................................................24 2.6 Synthesis of Hollow Gold Nanoshells .............................................................................26 2.6.1 Introduction .............................................................................................................26 2.6.2 Preparation of Hollow Gold Nanoshells ...................................................................27 2.6.3 Results .....................................................................................................................28 2.7 Discussion ......................................................................................................................31 Chapter 3. Cellular Uptake Studies of Gold Nanoparticles ..................................................33 3.1 Introduction ....................................................................................................................33 3.2 Materials and Methods ...................................................................................................35 3.2.1 Materials ..................................................................................................................35 3.2.2 Imaging of Gold Nanoparticle Cellular Uptake by Light scattering ..........................36 3.2.3 Nanoparticle Toxicity Test .......................................................................................37 3.3 Results............................................................................................................................38 3.3.1 Interpretation of Images ...........................................................................................38 3.3.2 Uptake of Gold Nanoshells ......................................................................................40 3.3.3 Uptake of Gold Nanorods ........................................................................................43 3.3.4 Uptake of Gold Core-Corona Nanoparticles .............................................................46 3.3.5 Uptake of Hollow Gold Nanoshells ..........................................................................47 3.3.6 Nanoparticle Toxicity Test .......................................................................................48 iv  3.4 Discussion ......................................................................................................................49 Chapter 4. Antisense Oligonucleotide Gene Therapy of Heat Shock Protein 27 .................51 4.1 Introduction ....................................................................................................................51 4.2 Materials and Methods ...................................................................................................54 4.2.1 Materials ..................................................................................................................54 4.2.2 ASO Transfection and Heating Procedure ................................................................54 4.2.3 ASO Concentration Test ..........................................................................................56 4.3 Results............................................................................................................................57 4.3.1 Hyperthermia of LNCAP and PC3 ...........................................................................57 4.3.2 ASO Transfection and Preliminary Heating Test ......................................................57 4.3.3 ASO Concentration Test ..........................................................................................58 4.3.4 ASO Transfection and Heating Experiments ............................................................60 4.4 Discussion ......................................................................................................................63 Chapter 5. Photothermal Treatment Studies using Gold Nanoparticles .............................65 5.1 Introduction ....................................................................................................................65 5.2 Materials and Methods ...................................................................................................68 5.2.1 Materials ..................................................................................................................68 5.2.2 Nanoparticle Heating Profiles ..................................................................................68 5.2.3 Photothermal In Vitro Heating Tests ........................................................................69 5.2.4 Photothermal In Vivo Heating Tests .........................................................................71 5.3 Results............................................................................................................................73 5.3.1 Nanoparticle Heating Profiles ..................................................................................73 5.3.2 Photothermal In Vitro Heating Tests ........................................................................75 5.3.3 Photothermal In Vivo Heating Tests: Preliminary Mouse Model Study ....................82 5.4 Discussion ......................................................................................................................85 Chapter 6. Summary and Future Work ................................................................................87 6.1 Summary ........................................................................................................................87 6.2 Future Work ...................................................................................................................88 References...............................................................................................................................90 Appendix ................................................................................................................................94 A.1 Gold Nanoshell Data ( > 50nm) .....................................................................................94 A.1.1 Synthesized Gold Nanoshells ..................................................................................94 A.1.2 Auroshell® Particles ...............................................................................................95 A.2 Images of Tumor Tissue ................................................................................................97 A.2.1 Light Scattering Images of Tumor Tissue ................................................................97 A.2.2 SEM-EDX Image of Tumor Tissue .........................................................................98  v  List of Tables Table 1. PEG types and coating concentrations. ........................................................................ 7 Table 2. Reactant volumes used for silica core synthesis. .........................................................11 Table 3. Typical incubation concentrations of gold nanoparticles for cellular uptake studies. ...36 Table 4. Gold nanoparticle incubation concentrations for toxicity assay. ..................................38 Table 5. Summary of gold nanoparticle uptake in prostate cancer cells. ....................................50 Table 6. Hyperthermia classifications [73]. ..............................................................................52 Table 7. Gold nanoparticle incubation concentrations for photothermal experiments. ...............69 Table 8. Test conditions for preliminary animal model experiments. ........................................71 Table 9. Summary of cell viability decreases and changes in temperature for photothermal experiments with 40nm gold nanoshells and Auroshell® particles in Figure 46. .......................80 Table 10. Summary of cell viability decreases and changes in temperature for photothermal experiments with 40nm gold nanoshells and 100nM ASO in Figure 47. ...................................81  vi  List of Figures Figure 1. Zonal anatomy of the prostate as described by McNeal [4]. Adapted from: [5]. .......... 1 Figure 2. Schematic of plasmon oscillation for a gold nanosphere. Adapted from: [14]. ............ 4 Figure 3. Illustration of a gold nanoshell. n1 = silica and n2 = gold. Adapted from: [13]. ............ 9 Figure 4. Schematic of nanoshell synthesis based on a seeded silica core. From: [28] with permission. ...............................................................................................................................10 Figure 5. TEM images of seeded 30nm silica cores (left) and complete 40nm gold nanoshells (right). ......................................................................................................................................13 Figure 6. Optical spectra of 40nm gold nanoshells at various stages of growth. ........................14 Figure 7. Size distribution data of silica cores (top) and bare 40nm gold nanoshells (bottom). ..15 Figure 8. Illustration of gold nanorod oscillation modes. Adapted from: [41]. ..........................17 Figure 9. TEM image of gold nanorods. ...................................................................................20 Figure 10. Optical spectra of CTAB and PEG 10000 coated gold nanorods. .............................21 Figure 11. TEM image of PEG 10000 coated Gold nanorods. ...................................................22 Figure 12. Illustration of a core-shell-corona particle. Adapted from: [38]. ...............................23 Figure 13. TEM image of gold core-corona nanoparticles. .......................................................25 Figure 14. Optical spectra of gold core-corona nanoparticles. ...................................................26 Figure 15. Schematic of hollow gold nanoshell synthesis. Adapted from: [49]..........................27 Figure 16. TEM image of hollow gold nanoshells. ...................................................................29 Figure 17. Optical spectra of hollow gold nanoshells. ...............................................................30 Figure 18. General schematic for endocytosis of gold nanoparticles in a cell. Source: generated using ChemBioDraw Ultra. ......................................................................................................34 Figure 19. Transmission (left) and scattering (right) images of control PC3 cells. .....................38 Figure 20. Transmission (left) and scattering (right) images of LNCaP incubated with 110nm Gold Nanoshells. ......................................................................................................................39 Figure 21. Cellular uptake comparison between LNCaP (left) and PC3 (right) with 40nm gold nanoshells. ...............................................................................................................................40 Figure 22. Cellular uptake comparison for 40nm gold nanoshells (left) and Auroshell® particles (right) in PC3 cells. ..................................................................................................................41 Figure 23. 3D scattering images of a single PC3 cell incubated with Auroshell® particles (left) and 40nm gold nanoshells (right). Arbitrary magnification. ......................................................42 Figure 24. Cellular uptake comparison of bare (left) and PEG coated (right) Auroshell® particles in PC3. .......................................................................................................................42 Figure 25. Microscope images of LNCaP with (right) and without (left) CTAB coated gold nanorods after a 24 hour incubation. Magnification 100X.........................................................44 Figure 26. Scattering images of a PC3 cells: with (right) and without gold nanorods (left). Arbitrary magnification. ...........................................................................................................44 Figure 27. Scattering image of LNCaP cells with PEG 10000 coated gold nanorods. Imaged at 800nm. .....................................................................................................................................45 Figure 28. Transmission (left) and scattering (right) images of LNCaP incubated with gold corecorona nanoparticles.................................................................................................................47 Figure 29. Cellular uptake of PC3 incubated with hollow gold nanoshells. ...............................48 Figure 30. Cytotoxicity profiles of LNCaP (left) and PC3 (right) incubated with bare and PEG coated 40nm gold nanoshells (NS40) and Auroshell® particles (NSAS). Concentrations outlined in Table 4. ................................................................................................................................49 vii  Figure 31. Proposed mechanism of stress-induced HSP synthesis in cells. Adapted from: [66]. Source: generated using ChemBioDraw Ultra ..........................................................................53 Figure 32. Hyperthermia profiles of LNCaP (left) and PC3 (right) cells at different temperatures and heating times. Cell viability by MTT assay. .......................................................................57 Figure 33. Preliminary transfection experiment on PC3 using 500nM ASO. Untreated (left) and transfected cells (right). Cell viability by trypan blue dye exclusion assay. ...............................58 Figure 34. ASO concentration test on PC3 (left) and LNCaP (right) at 37°C. Cell viability by MTS assay. ..............................................................................................................................59 Figure 35. Microscope images of untreated (left) and transfected (right) PC3 cells. Magnification 100X. ................................................................................................................60 Figure 36. Representative transfection and heating experiment on LNCaP with 100nM ASO. ASO transfected cell viabilities calculated with different AC values (Eq. 6). Untreated AC (top) and transfected AC (bottom) controls for ASO results only. Cell viability by MTT assay. .........61 Figure 37. Representative transfection and heating experiment on PC3 with 100nM ASO. Cell viability by MTT assay.............................................................................................................62 Figure 38. The NIR window. Adapted from: [15]. ....................................................................66 Figure 39. Illustration of a single pass experimental setup used for photothermal therapy of prostate cancer cells. Enlargement of one well in a 96 well plate. .............................................70 Figure 40. Thermocouple placement for skin (top) and tumor (bottom) test. Based on Table 8. 72 Figure 41. Temperature profiles for two concentrations (particles/mL) of gold nanorods at a laser power of 1W. ...................................................................................................................73 Figure 42. Temperature profiles for two concentrations (particles/mL) of 40nm gold nanoshells at a laser power of 1W. .............................................................................................................74 Figure 43. Representative photothermal experiment on PC3 incubated with 40nm gold nanoshells. Irradiated with a 4W laser with a 37C background temperature.............................76 Figure 44. Representative control experiment on LNCaP (left) and PC3 (right) without gold nanoparticles. Irradiated with a 4W laser with a 42 C background temperature. .......................77 Figure 45. Representative photothermal experiment on LNCaP (left) and PC3 (right) incubated with gold nanorods. Irradiated with a 4W laser with a 42 C background temperature. ..............78 Figure 46. Representative photothermal experiment on LNCaP (left) and PC3 (right) incubated with 40nm gold nanoshells (NS40) and Auroshell® particles (NSAS). Irradiated with a 4W laser with a 42C background temperature. .......................................................................................79 Figure 47. Representative photothermal experiment on LNCaP (left) and PC3 (right) treated with 100nM ASO and 40nm gold nanoshells. Irradiated with a 4W laser with a 42C background temperature. ..........................................................................................................81 Figure 48. Representative photothermal experiment on mouse model. Tested on healthy tissue. Irradiated with a 2W laser with and without direct injection of hollow gold nanoshells. ...........82 Figure 49. Representative photothermal experiment on mouse model. Tested on tumor tissue. Irradiated with a 1W laser with and without direct injection of hollow gold nanoshells. ...........83 Figure A1. Optical spectra of larger gold nanoshells based on 60nm and 90nm silica cores. .....94 Figure A2. TEM image of 90nm silica cores. ...........................................................................94 Figure A3. TEM image of 60nm seeded silica cores. ................................................................95 Figure A4. Size distribution data of Auroshell® particles, bare (top) and PEG coated (bottom) 95 Figure A5. Optical spectra of Auroshell® particles, bare and PEG coated. ...............................96 Figure A6. Transmission (left) and scattering (right) images of plain tumor tissue. ...................97 Figure A7. Transmission (left) and scattering (right) images of tumor tissue with hollow gold nanoshells injected. ..................................................................................................................97 Figure A8. SEM-EDX of tumor tissue with hollow gold nanoshells injected. ...........................98 viii  Acknowledgements I would like to express my deepest gratitude to my supervisor Dr. Ruth Signorell and cosupervisor Dr. Helen M. Burt for giving me the opportunity to study at the University of British Columbia and for the chance to work on an interdisciplinary project. This thesis would not have been possible without their guidance and support throughout my studies. I would also like to extend my appreciation to all of my committee members, Dr. Suzana K. Straus, Dr. Keng C. Chou and Dr. Bernard Shizgal for generously taking their time to review my thesis. I am indebted to Dr. Sherry Wu, who has been a very valuable guide throughout this project. She taught me everything I needed to know on cell culture and cell-based assays, and always shared her experience and expertise without any reservation. There were many valuable discussions, especially regarding results presented in several chapters in this thesis. I would like to thank Dr. Qifeng Li for his assistance with imaging, using light scattering microscopy, and the photothermal experiments. This research would have not been fruitful without his contribution. I also greatly thank Thomas Preston for his help in getting me started with the gold nanoparticle synthesis and his useful comments on some thesis chapters. Many thanks go to Dr. John Jackson for his encouragement, and all the in-depth knowledge he shared during this study. This thesis could not have been completed without the support of Dr. Martin Gleave (Prostate Center) who supplied many valuable materials used in this project. Dr. Saeid Kamal (LASIR) and Dr. Elena Polishchuk (UBC Bioservices) were gracious in providing the laboratory facilities necessary for this research. I am thankful to the staff of the UBC BioImaging Facility (Garnet Martens and Derrick Horne) for their assistance with imaging nanoparticles. I would also like to thank all those I had the privilege to work with over the past two years at the University of British Columbia, in particular all current and former members of the Signorell and Burt research groups. I truly enjoyed working with all of you. Lastly, I would like to thank my family and friends for all their love and encouragement. I am especially grateful to Matthew Bacque and, my father, Yee Leung for all their extra support. I also want to thank all the wonderful people I have met in Vancouver. They provided inspiration, valuable advice and made my time in Vancouver really wonderful. I value all your friendship and support.  ix  Chapter 1. Introduction Nanotechnology is a rapidly growing field that is at the forefront of many scientific advancements and new technology. In particular, the use of nanomaterials in medicine is of interest as there is a great potential to improve existing or develop new treatment methods for disease. Cancer is one of the major causes of mortality in industrialized countries and its incidence is increasing [1]. Current treatments available for cancer are often quite invasive, can result in damaging healthy tissue and/or are unable to completely destroy cancer leading to recurrences. Therefore, exploring and developing alternative therapies that can improve treatment are important. 1.1 Prostate Cancer Cancer is a disease in which a group of cells display uncontrolled growth, invasion and sometimes metastasis [1]. Prostate cancer starts in the cells of the prostate gland which is part of the male reproductive system. The prostate lies at a critical juncture between the bladder, urinary sphincter, rectum and pubis which makes treatment of cancer in this location difficult, as there is high risk of damaging surrounding organs [2]. Prostate cancer is the most common cancer in Canadian men as it represents 27% of diagnosed cancers. Statistically, there is a 1 in 7 chance of developing it and a 1 in 27 chance of dying from it [3].  FIGURE 1. ZONAL ANATOMY OF THE PROSTATE AS DESCRIBED BY MCNEAL [4]. ADAPTED FROM: [5].  The prostate has three anatomic zones to which a tumor can be located (Figure 1). The transition zone represents about 5 to 10% of the glandular tissue and the central zone forms part of the base of the prostate. The bulk of the prostate is constituted by the peripheral zone [5]. The 1  relative number of prostate cancers that arise in the central zone, peripheral zone and transition zone are approximately 10, 70 and 20% [4]. Prostate cancer tumors often grow slowly and are considered clinically insignificant if they are confined within the gland, are well-differentiated and/or are small in size (< 0.5 cm) [6]. However, some prostate cancer tumors can become aggressive and can spread, so screening and monitoring is important. Widespread screening for prostate cancer is done by determining prostate-specific antigen (PSA) levels. The detection of elevated PSA values can help estimate tumor volume and stage, and prompt further investigation by transrectal ultrasound, digital rectal examination or prostate biopsy [6]. Information from these will classify the risk of the tumor using the Gleason score and clinical stage, which will determine if treatment is necessary. 1.2 Prostate Cancer Treatment Modalities Treatment of prostate cancer depends on the patient and the assessment of their risk factors, as there is not a uniform set of treatment criteria for all stages of the disease. For those who have localized prostate cancer, the biological threat will determine if the tumor can be safely observed or if treatment is required. In general, the more advanced the disease the greater the need for therapy. Although not well validated, many physicians believe that tumor sizes > 0.5cm are clinically significant [6]. For tumors requiring treatment, the therapy choice will depend on the stage of the disease, location and if metastases is present [2]. Major treatment modalities for localized prostate cancer are radiation (external beam or brachytherapy), androgen-deprivation hormone therapy, surgery/prostatectomy and chemotherapy. Although most of these therapies have shown treatment success, all of these therapies have demonstrated cancer recurrence [2, 7] and/or are invasive. For example, the side effects of surgery and radiation therapy include problems with urinary, bowel and sexual function [6]. Alternative therapies, such as cryotherapy and high intensity ultrasound (HIFU) have shown promise, but have difficulty treating large tumor volumes and can also damage bodily functions [8]. Prostate cancer treatment often combines many of these therapies, either as a cooperative treatment or sequentially as an initial and salvage therapy (cancer recurrence). However, a majority of these cancer treatments lead to adverse effects and negative consequences on quality of life. This leads to considerable interest in developing a method of treatment that is less invasive with the capability to completely cure or control prostate cancer.  2  1.3 Project Purpose The overall research proposal was based on a joint collaboration between the Signorell group (Department of Chemistry) and Burt group (Department of Pharmaceutical Sciences) UBC, Canada, and the Gleave Group (Prostate Center) VGH, BC, Canada to develop a multimodal therapeutic strategy based on photothermal therapy using gold nanoparticles, chemotherapy drugs (paclitaxel and/or docetaxel) and gene therapy, using antisense oligonucleotides (ASO) targeting HSP27 and clusterin, for localized prostate cancer treatment. A formulation of these components would be injected directly into the tumor, in order to destroy cancer cells using the effect of each component. Gold nanoparticles were selected for their photothermal properties, as they can induce hyperthermia when combined with laser irradiation. However, they also have the potential to act as a drug delivery agent. Taxane drugs were selected as they are the most active chemotherapy agents against prostate cancer [9]. Treatment with ASO was added to sensitize the cancer cells to heat and taxane drug treatment. It was hypothesized that the thermal ablation of prostate cancer tumors could be performed at noninvasive or minimally invasive temperatures using this multimodal approach. Co-delivery of taxane drugs and ASO would supplement and/or support cancer cell death, leading to complete tumor destruction. These conditions combined with direct injection into the tumor could prospectively minimize damage to surrounding organs. Several project objectives were obtained from this original proposal for investigation in this present study. An objective was to synthesize and characterize gold nanoparticles that could induce hyperthermia under irradiation at near infrared (NIR) frequencies. Target properties of the gold nanoparticles were absorption in the NIR, size < 100nm, good cellular uptake and a simple synthesis. In vitro and in vivo prostate cancer model studies were used to measure cellular uptake and/or the effectiveness of photothermal therapy using the gold nanoparticles and laser irradiation. ASO gene therapy, targeting HSP27, was investigated to examine if the cancer cells could be sensitized to heat treatment in order to increase cell death at lower temperatures.  3  Chapter 2. Gold Nanoparticle Synthesis and Characterization 2.1 Introduction Interest in gold nanostructures has increased dramatically because of their potential applications in many disciplines. Their unique properties, such as biocompatibility, modifiable particle surface and strong tunable photoabsorption [10], allow gold nanoparticles to be easily manipulated, so that different concepts and approaches may be explored. There are four main classifications for applications: labeling, delivering, heating and sensing [11]. In this work, gold nanoparticles with the appropriate optical properties, size and stability for photothermal therapy were selected, and evaluated on how simple and feasible they were for process scale-up. The nanoscale dimensions of gold results in the observation of new properties possessed neither by the metal or atoms forming the metal [12]. The interaction of light with a gold nanoparticle results in a collective coherent oscillation of the conduction electrons (Figure 2) that resonates at a particular frequency known as the surface plasmon resonance (SPR). This resonance gives nanoparticles unique optical properties, such as the enhancement of absorption and scattering. For smaller nanoparticles, absorption dominates over scattering, whereas for larger nanoparticles scattering dominates over absorption [13].  FIGURE 2. SCHEMATIC OF PLASMON OSCILLATION FOR A GOLD NANOSPHERE. ADAPTED FROM: [14].  The SPR is influenced by nanoparticle size, shape, aggregation and environment. Each factor can be used to tune the wavelength and intensity of the SPR band, allowing for specific regions of the visible and near infrared (NIR) spectrum to be targeted. However, in practice only some types of nanoparticles are suitable for certain applications. For example, nanoshells and nanorods can be tuned for NIR radiation, whereas pure gold nanospheres cannot [13]. The 4  requirements for photothermal applications are that the nanoparticles must be optically and thermally robust, and absorb in the NIR (650 to 900nm) [15] as there is minimum light absorption by tissue in this region [16]. In photothermal therapy, the nonradiative processes of gold nanoparticles are exploited. Light is absorbed at SPR wavelengths, exciting the coherent motion of electrons. This leads to relaxation and conversion of the electromagnetic radiation to heat. Heat diffusion to the surroundings occurs quickly and is efficient at low photon densities. [17] The appeal of gold nanoparticles as photothermal agents is that the majority of light absorbed is converted to heat [17, 18]. In general, mammalian cells internalize gold nanoparticles by specific or nonspecific routes [11]. Specific uptake requires the particle surface to be modified with ligands that recognize receptors on the cell membrane. For nonspecific routes, particles are taken up by natural cell processes, such as endocytosis. Particle sizes between 20 to 100nm preferentially accumulate at tumor sites [1] with particles under 50nm exhibiting the best cellular uptake [19]. For nonspecific uptake, small sizes allow for easy passage through cell membranes (in vitro) and can avoid any negative side effects (in vivo) [20-22]. Uptake by either method requires nanoparticles to be stable and disperse under physiological conditions. The synthesis and characterization of gold nanoparticles of various shapes and sizes has been well documented in the literature [14, 21], leading to numerous synthesis/fabrication pathways and many standard techniques to analyze their properties. In general, gold nanoparticles are produced by reduction of a water soluble gold salt, such as HAuCl4. Stabilizing agents can be either adsorbed (e.g. surfactant) or chemically bound (e.g. polymers), to help maintain colloidal stability [11]. While there are various coatings available, polyethylene glycol (PEG) is often used to provide stability, reduce nonspecific adsorption on the surface [11], increase biodistribution [16] and suppress the immune response [22]. This gives PEG coated gold nanoparticles a “stealth property”, as it can improve efficiency for in vivo delivery by increasing circulation time in the blood [16, 23]. Building a PEG monolayer on gold nanoparticles is easily done as the surface can be functionalized with sulfur or amine moieties, which form a covalent bond with gold. Incubation with a solution of PEG often results in an instantaneous and complete coating. Four nanoparticle candidates were chosen for this study, gold nanoshells, gold nanorods, gold core-corona nanoparticles and hollow gold nanoshells. These particles have the potential to meet all the required properties, such as a size below 100nm to ensure good cellular uptake and 5  to maximize absorption/heat dissipation. Absorption at ~800nm was chosen for the target wavelength in the NIR window. 2.2 Materials and Methods 2.2.1 Materials Tetrachloroauric(III) acid (HAuCl4 H2O, ≥ 99.9%), tetraethylorthosilicate (TEOS, 98%), tetrakis(hydroxymethyl)phosphonium chloride (THPC, 80%), 3-aminopropyltrimethoxysilane (APS, 97%), hexadecyltrimethylammonium bromide (CTAB, 99%), silver nitrate (AgNO3, 99%), L-ascorbic acid (C6H8O6), potassium carbonate (K2CO3, ≥ 99%), polyvinylpyrrolidone (PVP, MW = 55kDa ), trisodium citrate dihydrate (Na3C6H5O7·2H2O), and ammonium hydroxide (NH4OH, 28-30%) were obtained from Sigma-Aldrich. Sodium borohydride (NaBH4, 98%), sodium hydroxide (NaOH, 99.8%), hydrochloric acid (HCl, 36.5-38%), nitric acid (HNO3, 65-70%) and formaldehyde (H2CO, 30%) were obtained from Fisher Scientific. Thiol-terminated polyethylene glycol (mPEG-SH, MW = 2000, 10000 and 20000Da) were obtained from Laysan Bio Inc.. All chemicals were used as received. Distilled water was used in all the experiments. Water was sterilized as needed by filtering through a 0.22µm filter. Reactions were performed in polypropylene containers unless otherwise indicated. Glassware used was cleaned by aqua regia and rinsed with distilled water. The synthesis of the different gold nanoparticles is explained in Sections 2.3 to 2.6. Commercially prepared bare (uncoated) and PEG coated (MW= 5000) Auroshell® particles were obtained from Nanospectra Biosciences Inc.. These particles are 150nm gold nanoshells with a silica core of 120nm and a gold shell thickness of 15nm. Concentrations of the particles, determined by Mie theory by the manufacturer, were 2.894 x 109 and 2.828 x 109particles/mL for bare and PEG coated respectively. The size of the Auroshells® was measured using DLS (Appendix A.1.2). These particles were used in Chapter 3 to determine cellular uptake and in Chapter 5 as a standard for comparison for photothermal therapy. 2.2.2 PEGylation Gold nanoparticles were PEGylated in an excess amount of PEG. All PEG solutions (Table 1) were prepared in sterile water. The gold nanoparticle solution (0.5mL) was added to PEG solution (0.5mL). Particle concentrations were adjusted by monitoring absorbance 6  and were typically in the range outlined in Table 3. The mixture was stirred overnight at room temperature and purified by either centrifugation or dialysis. TABLE 1. PEG TYPES AND COATING CONCENTRATIONS.  Polymer PEG 2000 PEG 10000 PEG 20000  Molecular Weight (kDa) 2 10 20  Concentration (mg/mL) 1.0 5.0 10.0  Approximate Coating Thickness (nm) [23] ≤5 5-10 10-20  Coating with PEG was used to evaluate the effect on gold nanoparticle stability and cellular uptake. Niidome et al. showed that PEG chains between 2 and 20 kDa can stabilize nanoparticles [23], therefore this range was used for coatings. In general, a longer chain length leads to a thicker PEG coating, which adds to the overall particle size. 2.2.3 Methods of Purification Centrifugation/Redispersion Aliquots of gold nanoparticle solutions (1mL) were placed in 1.5mL microfuge tubes and typically centrifuged at a desired speed (rpm) and time length (minutes), depending on the size and type of particle. The supernatant was removed leaving the pellet intact. Water was added and the pellet was redispersed by vortex or sonication. The process was repeated at least twice.  Dialysis Aliquots of the gold nanoparticle solutions (1 to 2mL) were placed in dialysis tubing (Spectra/Por) at room temperature against ~2L of water which was changed periodically four times and left to stir overnight. 2.2.4 Characterization UV-vis Spectroscopy Spectra were recorded on a Varian 50 Bio UV-visible spectrophotometer over the range from 400 to 1100nm, using a 10mm path-length quartz cuvette (Hellma). UV-vis spectroscopy is the most common method of characterizing nanoparticles, as the absorption relates to the properties of metal nanoparticles, such as size, shape and aspect ratio. 7  Dynamic Light Scattering (DLS) and Zeta Potential Nanoparticle size and zeta potential was measured with either a Malvern Zetasizer 3000 HS or a Malvern Zetasizer Nano-ZS. All samples were dispersed in water and held in disposable cuvettes.  Transmission Electron Microscopy (TEM) Electron micrographs were recorded to determine the size and the shape of the gold nanoparticles. Measurements were done on a Hitachi H7600 Transmission Electron Microscope at an operating voltage of 80kV. Samples were prepared by placing the sample solution (10µL) onto a Formvar/carbon coated, 200 mesh copper grid (Ted Pella Inc.) The sample grid was allowed to dry by evaporation. For TEM imaging of PEG coated particles, observation of the coating may be enhanced by staining the TEM slide with uranyl acetate. One drop of the uranyl acetate (1%, ~5µL) was added to the sample on the TEM grid and removed after one minute with filter paper.  Inductively Coupled Plasma Mass Spectrometry (ICP-MS) The gold nanoparticle concentration can be calculated by determining the total gold content by ICP-MS. A typical digestion procedure was done by mixing gold nanoparticle solution (0.5 to 1.0mL) with aqua regia (1:3 volume ratio of HNO3:HCl, 2.5mL) and reacting for 2 hours [24]. The samples were then diluted to 50mL in water. The ICP-MS analysis was performed by ALS Enviro, BC, Canada. The following equations may be used to convert the number of gold atoms to spherical gold nanoparticles [19]: U = 2/3π (D/a)3  (Eq. 1)  N= M/U  (Eq. 2)  U is the number of gold atoms per particle, D is the diameter of the particle, a refers to the edge of unit cell (4.0786Å), M is total number of gold atoms per mL and N is the number of particles per mL. Particles consisting of a gold shell, the volume/atoms of the core were subtracted.  8  2.3 Synthesis of Gold Nanoshells 2.3.1 Introduction Nanoshells are a special class of nanocomposite materials that consist of a core covered by a thin shell (Figure 3). They are highly functional particles with properties that can be tailored by changing the composite materials or the core-to-shell ratio [25]. Although nanoshells can be synthesized in a variety of combinations using dielectric, semiconductor and metal materials, the particle combination of interest was a dielectric silica core (n1) with a thin gold coating (n2). Gold nanoshells are typically synthesized with diameters ranging from 80 to 150nm [22], however smaller and larger sizes are also possible. The position of the extinction peak can be tuned from 600 to 1000nm and the relative contributions of absorption and scattering can be configured by changing the core-to-shell ratio, i.e. the size of the core (r1) divided by the shell thickness (r2 minus r1) [13].  FIGURE 3. ILLUSTRATION OF A GOLD NANOSHELL. N 1 = SILICA AND N 2 = GOLD. ADAPTED FROM: [13].  Large nanoshells ( > 100nm) tuned to absorb NIR radiation have been studied for use in photothermal applications, and exhibit efficient conversion of absorbed radiation into heat and are thermally robust at therapeutic temperatures [13]. Although these larger particles have good heating behavior, their size is not optimized for this purpose. Larger particles scatter a larger fraction of light and require more energy to heat the nanoshell, as there is a greater mass of gold present leading to less heat dissipation to the surroundings [26]. Smaller nanoshells are more attractive because absorption is maximized and a greater temperature 9  rise is possible due to the smaller mass of gold. Nanoshells < 100nm in size, have been studied far less, with only a few reports of nanoshells with diameters of 10 to 60nm being synthesized [22] and only one report of a photothermal application [27]. As mentioned previously in Section 2.1, small particles under 50nm in diameter have better uptake into cells, so further research into smaller nanoshells is desirable.  FIGURE 4. SCHEMATIC OF NANOSHELL SYNTHESIS BASED ON A SEEDED SILICA CORE. FROM: [28] WITH PERMISSION.  There are several different synthetic pathways for the production of gold nanoshells, each with varying complexity and quality of product. However, the majority of pathways follow the general scheme shown in Figure 4: synthesis of the silica core, functionalization of the core with amines, seeding with gold and growth of the shell. Overall, most methods can be modified to produce different sizes by changing amount or type of reactant(s). Although the method selected in this work is traditionally the most common route for gold nanoshells, smaller nanoshells (≤ 50nm) have not been previously synthesized. However, the procedure is well developed and has known chemistry that can be manipulated. The fabrication of nanoshells requires several steps, but each is simple and straightforward. The only limitation is the numerous purification steps. These particles are of economic interest, as the bulk of the particle consists of an inexpensive material (silica) [25]. 2.3.2 Preparation of Gold Nanoshells Synthesis of Gold Colloids Small gold particles were formed from the reduction of gold salt by strong reducing agents. Gold particles of < 3nm in diameter were synthesized by a method described by Duff et al [29]. In 45mL water, NaOH (0.2M, 1.5mL) and THPC solution (0.8% in water, 1mL) was added. Under rapid stirring, an aqueous solution of HAuCl4 (1%, 1.5mL) was added producing a clear, orange-brown color. The solution was moderately stirred for 10 minutes, 10  stored in the dark at 4C, and aged at least one day before use. Prior to use, the particles were analyzed by UV-vis. Size was confirmed by TEM.  Synthesis and Amination of Silica Cores Silica nanoparticles with a diameter of 30, 60 and 90nm were synthesized by a modified Stöber method [30, 31]. The cores form by means of hydrolysis of alkyl silicates and subsequent condensation. Therefore, the size can be controlled by the concentration of ammonia and water. All reactant volumes for the synthesis, for different core sizes, are given in Table 2. In 50mL of dry ethanol, ammonium hydroxide (28-30%) was added. Under rapid stirring TEOS (98%) was added and then stirred at 100rpm for 8 hours, resulting in a slight opaqueness. As there is a weak interaction between silica and gold, the silica surface must be modified with a functional group, such as an amine. This gives silica a nucleophillic surface for adsorption of colloidal gold [32]. Amination was done by adding APS (97%) in excess, to ensure a complete coating, and stirred overnight. The cores were centrifuged (3500rpm) for 30 minutes and redispersed in ethanol. The size was determined by DLS and TEM, and amination was confirmed by measuring the zeta potential. TABLE 2. REACTANT VOLUMES USED FOR SILICA CORE SYNTHESIS.  Silica Core Size (nm) 30 60 90  Ammonium Hydroxide (mL) 1.5 2.0 3.0  TEOS (mL)  APS (µL)  1.5 1.5 1.5  24.5 15.8 20.0  Several different core sizes were produced in preliminary studies. However, the 30nm core particles were the main focus, as they produce particles that satisfy the optimal size criterion for cellular uptake. Based on the average particle radius (r), the approximate concentration in particles/mL can be calculated using the total volume (v), the molecular weight of silica (MWSiO2), 2g/cm3 [33] as the density of silica (ρSiO2) and nSi(OC2H5)4 the total moles of TEOS, assuming all TEOS forms spherical particles of the prescribed size [34]. The concentration of silica particles can be expressed as: [Silica NPs] = (nSi(OC2H5)4)(MWSiO2) 4/3πρSiO2r3v  (Eq. 3)  11  Seeding of Cores with Colloidal Gold The surface of the silica particles was seeded with colloidal gold so additional gold could be plated to form a continuous layer on the core. The method by Oldenburg et al. was used to seed the cores [35]. Aminated silica nanoparticles (2 x 1014particles/mL, 100-200µL) were added to a solution of gold colloid (2 x 1015particles/mL, 5mL). The solution was vortexed and left to stand overnight at room temperature in the dark. Non-attached gold particles were removed by centrifuging (3600 rpm) for 30 minutes and redispersing the pellet in water twice. By the second centrifugation/redispersion step the supernatant contained negligible amounts of free gold. The seed coverage was confirmed by TEM.  Growth of Gold Nanoshells The gold shell was completed by the reduction of gold. A modified method by Rosenthal et al. was used [36]. Gold solution used to grow the shell was prepared at least 16 hours in advance by dissolving K2CO3 (100mg) in water, adding HAuCl4 (1%, 6mL) and adding water to a final volume of 100mL. The solution appeared yellow initially and slowly became colorless. The coating was done by diluting different amounts of the gold solution in water to a final volume of 4mL, and adding the seeded silica cores (200µL). Under mixing (~240rpm) formaldehyde (30%, 10µL) was added. The solution became colored, ranging from blue/purple/pink, after 20 minutes. The reaction was monitored by measuring the UV-vis absorption for up to 4 hours of stirring. This process forms a highly crystalline gold shell through Ostwald ripening [13]. The UV-vis absorption, of the final product, was measured and TEM and DLS confirmed the size. Particles that exhibited the correct properties (e.g. NIR absorption) were centrifuged (14000rpm) for 30 minutes and redispersed in sterile water. The concentration of nanoshells was determined by taking into account the various dilution steps used on the silica core concentration and assuming all cores were coated. 2.3.3 Results Silica cores of all sizes were successfully synthesized with a reasonable size distribution and spherical shape. Characterization was focused on the 30nm diameter core as these met the target size of < 100nm for the nanoshell. An average core diameter of 30nm was measured by DLS and TEM. Amination of the silica cores was confirmed by a positive zeta potential (+35.5mV), which indicated a positive surface charge. The TEM images of seeded 12  silica cores (Figure 5) show adequate coverage of the cores by the gold seeds. The nanoshells produced have a shell thickness of 5 to 10nm and a final nanoshell diameter of 40 ± 11nm (TEM and DLS), producing a core-to-shell ratio of 3 to 1.5. The zeta potential of the particles became negative when the shell was complete. This occurs because anions adsorb to the gold surface giving the particle an overall negative charge. The calculated concentrations by Eq. 3 of silica cores and nanoshells for a typical synthesis were 2.3 x 1014 and 2.2 x 1011particles/mL, respectively. However, the concentration of nanoshells may be overestimated by this method, as there are expected particle losses during purification steps. The nanoshell concentration determined by ICP-MS was 8.6 x 1013particles/mL, which was slightly higher than the value determined by Eq. 3. Both these values were considered to be good approximations, as the final nanoshell concentrations were in relatively good agreement. Both concentration determinations are limited by two assumptions: the reaction produces perfect spheres with uniform shell thicknesses and by-products are not formed.  FIGURE 5. TEM IMAGES OF SEEDED 30NM SILICA CORES (LEFT) AND COMPLETE 40NM GOLD NANOSHELLS (RIGHT).  Calculations based on Mie theory, requires a core-to-shell ratio of 7 to generate a plasmon peak of 790nm for a 30nm diameter silica core [22]. This means a 2nm shell thickness was required. However, in these calculations the particles were also assumed to be uniform, spherical and smooth. Although the shell thickness generated in this work was at minimum 5nm, and not 2nm, the small nanoshells do exhibit a tail-end absorption in the NIR, rather than an absorption maximum. The growth of the 40nm gold nanoshell (Figure 6) was very consistent with the optical trends observed experimentally by Rasch et al. for the growth 13  of nanoshells on 28nm and 38nm cores [22].The NIR portion of the extinction increases, as the shell nears completion (A  C in Figure 6) leading to a broad peak. This broadness and deviation, from expected optical behavior, for the given core-to-shell ratio was attributed to surface roughness, non-spherical particle shape, distribution in size and aggregation [22].  FIGURE 6. OPTICAL SPECTRA OF 40NM GOLD NANOSHELLS AT VARIOUS STAGES OF GROWTH.  Aggregation of particles results in an increase in the overall plasmon resonance wavelength, as the plasmons of particles interact [37]. Although, Rasch et al. reports aggregation as a contributing factor to the shape of the extinction peak [22], the size distribution of produced nanoshells show a single peak for silica cores and complete nanoshells (Figure 7). This result suggests fairly disperse solutions for bare particles. If there was aggregation, it is expected to have minimal contribution to the observed extinction in Figure 6. However, aggregation may be prevented, especially during periods of long-term storage of solutions, by coating with PEG. The PEG coating of nanoshells was not expected to be uniform on all spots on the surface. For example, the layer was estimated by DLS to be 5 to 10nm thick for a PEG 10000 coating. PEG coated and bare particles have similar extinction spectra as the resonance is broad and cannot exhibit shifting (e.g. red or blue shift). The only noticeable difference was that the PEG coated particles were more difficult to purify by centrifugation, as the particles were very disperse. This required PEG coated 14  particles to be purified by dialysis. The MW of PEG did not seem to significantly change the stability of the particles synthesized. The size distribution (Figure 7) for both the cores and nanoshell was reasonable, even though the particles were less spherical. The 30nm silica cores were more spherical than the finished nanoshell (Figure 5). Loss in uniform shape was attributed to the gold seeds, rather than the size of the silica cores. Variation in size distribution of the seeds and any clustering at the silica surface is expected to give a rough and uneven surface [22]. This roughness becomes more prevalent when coating small silica cores or when generating thin gold shells.  FIGURE 7. SIZE DISTRIBUTION DATA OF SILICA CORES (TOP) AND BARE 40NM GOLD NANOSHELLS (BOTTOM).  Typically, larger nanoshells have been favored because of their more uniform properties, such as spherical shape and smoother surfaces, leading to a tunable plasmon peak. However, for photothermal applications, it has not been determined whether these characteristics are necessary and/or more favorable. Shape and crystallographic facets are important for certain applications, such as catalysis or applications dependent on surface activity [12]. In other cases, nanometer scale roughness has been shown to enhance the optical properties of gold nanoparticles [13, 38, 39]. In this work, surface roughness acts to expand the absorbance window into the NIR for small nanoshells shown in Figure 6. The extinction of small 15  nanoshells was expected to be influenced by surface roughness, non-uniform shape, distribution in size and aggregation. While, the degree of influence for each factor is unknown, the net effect creates particles with a broad extinction that includes the NIR. Gold nanoshells based on the 60 and 90nm silica cores were also synthesized. The cores were more spherical compared to the 30nm cores. The shell thickness for the 60nm cores was estimated by DLS or TEM to be 10 to 15nm and for the 90nm cores 10 to 20nm, leading to total nanoshell diameters of 80 to 90nm and 110 to 130nm, respectively. The optical spectra of the complete nanoshells exhibit a broad absorption with a tail end in the NIR. It was assumed that the broad extinction is attributed to the same factors as the smaller 40nm gold nanoshells. Characterization data for these larger nanoshells are in Appendix A.1.1. The Auroshell® particles from Nanospectra Biosciences Inc. have an extinction spectrum that has a maximum in the NIR, as these particles are large enough that the absorption band may be tuned by the core-to-shell ratio. Concentration values for the particles were given as 2.894 x 109 and 2.828 x 109particles/mL for bare and PEG coated particles. However, the concentration determined by ICP-MS analysis gives a value of 6.1 x 1012particles/mL for the bare particles. This variation may be attributed to the different methods in determining concentration and the associated factors as previously discussed. The optical spectra and DLS data for these particles can be found in Appendix A.1.2. The synthesis of nanoshells consists of a few simple steps. However, some reaction steps can be laborious. Nevertheless, the desirable properties of the nanoshell for several applications drive research that leads to shortening and/or simplifying the synthesis further. Nanoshells have tunable optical properties and are thermally robust which makes them appropriate for photothermal therapy. Work by Kah et al. simplified the synthesis by using a single step deposition-precipitation process to seed several sizes of silica cores (80 to 440nm) with gold hydroxide, removing the separate synthesis of colloidal gold [32]. Storti et al. further shortened the synthesis by developing a one-pot synthesis of nanoshells with diameters below 30nm [27]. Therefore, scaling of the total synthesis is possible. Another attractive factor is the bulk of the nanoshell particle volume consists of inexpensive silica [25]. Further improvements to the synthesis could also be done during the seeding step. If highly stable gold seeds under 1nm could be produced, a thinner and more uniform shell may be possible.  16  2.4 Synthesis of Gold Nanorods 2.4.1 Introduction Gold nanorods are a distinctive class of nanostructures that have highly shape dependant optical properties. Rod-shaped nanoparticles show two absorption bands in their spectra that correspond to longitudinal and transverse surface plasmons modes (Figure 8). The longitudinal mode is a strong, long wavelength band due to the oscillation of the conduction band electrons along the longer axis, while the transverse mode is a weaker, short wavelength band that is a result of oscillation along the shorter axis [40]. When tuning the absorption of nanorods, the aspect ratio (length/width) is manipulated usually by changing rod length. The rods are tuned so the transverse band is in the visible region and the longitudinal band is shifted within the NIR region. Usually aspect ratios of 1.5 to 10 result in the longitudinal absorption maxima between 600 and 1300nm [12]. The dimensions of the rods can be synthetically controlled and the longest axis can be kept under 80nm to achieve an absorption in the NIR. Gold nanorods are attractive for photothermal applications, as they have a highly efficient absorption and a larger absorption cross-section per unit volume than most other nanostructures [17].  FIGURE 8. ILLUSTRATION OF GOLD NANOROD OSCILLATION MODES. ADAPTED FROM: [41].  Nanorods may be synthesized by two main methods, rigid templates or surfactants [42]. There are numerous methods for each type, which produces nanorods of varying specifications and quality. The most common wet chemical synthesis is a seeded growth method using the cationic surfactant, CTAB. This surfactant allows for more size and shape control and has the most reproducible synthesis [42]. The growth mechanism is not completely understood, but is proposed to involve the formation of CTAB micelles. The 17  cationic nature of CTAB makes coated nanorods, in principle, an ideal candidate for cellular uptake as positively coated particles have higher uptake [19]. However, there has been some question to the cytotoxicity of nanorods coated with CTAB. Some studies suggest that CTAB stabilized nanorods do not effect cell viability, while others have demonstrated cytotoxicity [13]. Common practices for removing CTAB are surfactant exchange or displacement by polymers, such as PEG. 2.4.2 Preparation of Gold Nanorods The nanorod synthesis was adapted from the seed-mediated growth method by Nikoobakht and El-Sayed [42]. All reactions were performed in glass vials.  Preparation of Seed Solution A solution of CTAB (0.20M) in water was prepared by gently warming the solution. In CTAB solution (0.20M, 5mL), water (4.9mL) and an aqueous solution of HAuCl4 (1%, 100µL) were added and stirred. To this ice cold NaBH4 (0.01M, 0.6mL) was added and the solution was vigorously stirred for 2 minutes. The solution changed from colorless to a light brown, as surfactant capped seeds of less than 4nm in diameter were formed. The solution was kept at 25°C to prevent the precipitation of CTAB.  Preparation of Growth Solution To CTAB (0.20M, 5mL) differing volumes (0.15, 0.20, 0.25 and 0.30 mL) of AgNO3 (4.0mM) was added, as increased amounts of silver red shifts the longitudinal plasmon band of the nanorods. Under moderate stirring, water (4.8mL) and HAuCl4 (1%, 200µL) were added, followed by ascorbic acid (78.8mM, 70µL). The color of the growth solution changed from deep yellow to colorless. The solution was maintained at a temperature of 25 to 30 °C.  Growth of Gold Nanorods The seed solution (12µL) was added to the growth solutions at 25 to 30 °C, and stirred moderately for 5 minutes and then left stationary. Each solution gradually changed color after about 20 minutes. Longer nanorods required more time, indicated by the slow color change. The UV-vis absorption was measured to determine the position of the longitudinal maxima and TEM was used to determine the shape and size. Particles that exhibited the desired 18  properties were centrifuged (10,000rpm) and redispersed in sterile water twice. The concentration of nanorods may be determined by using the amount of reactants as before for the silica cores in nanoshells (Section 2.3.2), except using the formula for a spherically capped cylinder. However, due to the presence of other smaller shapes and weak reducing conditions, only about 15% of gold forms rods [43]. Concentration was determined by another method, using the measured absorbance. Orendorff and Murphy calculated the molar extinction coefficients () at the longitudinal plasmon maxima () of nanorods with aspect ratios of 2.0 to 4.5 [43]. The relationship was found to be approximately linear over the given range. The corresponding equation from their work is:  = 1 x 107  - 5 x 109  (Eq. 4)  Once the extinction coefficient (M-1cm-1) was calculated, the concentration in moles of nanorods per L [43] was calculated using the Beer-Lambert Law, with A as the absorbance of the sample and 1cm as the path length (l). The concentration was converted into particles/mL using Avogadro’s number. [NR] = 6.02 × 1023 A l  (Eq. 5)  Gold Nanorod PEGylation PEGylation of gold nanorods was done by a modified procedure of Liao and Hafner [44]. The twice centrifuged/redispersed nanorods (~9 x 1010particles/mL, 0.5mL) and PEG solution (0.5mL) (see Table 1) were mixed with potassium carbonate (2mM, 100µL). The mixture was stirred overnight at room temperature and purified by dialysis. The UV-vis absorption and zeta potential was measured to confirm a successful coating. 2.4.3 Results The absorption of nanorods can be easily tuned to NIR wavelengths. The synthesis selected, varies the amount of silver to control the length of the nanorods during the growth step. Nanorods with an absorption maximum at ~800nm had an aspect ratio of 3.5 to 4.0, with a length of 73 ± 7nm and width 21 ± 2nm (Figure 9). This closely matches results published by Jain et al [45]. Examination of TEM images for several syntheses, returned at minimum 95% of rod-shaped particles, with the remaining 5% as cubes, spheres and other shapes.  19  FIGURE 9. TEM IMAGE OF GOLD NANORODS.  There have been several insights into the mechanism of rod formation in literature. A proposed mechanism is that the surfactant forms a soft template and growth is controlled by the surfactant concentration and ionic strength [42]. The role of silver, in the synthesis used in this present study, is rationalized by Orendorff and Murphy. They proposed silver deposits faster on the sides of the rods leading to inhibition of growth on the sides and preferential growth at the ends [43]. Therefore, increasing silver content allows more silver deposition on the sides leading to longer rods.  20  0.6  0.5  Absorbance  0.4  NR NR PEG  0.3  0.2  0.1  0 400  500  600  700  800  900  1000  1100  Wavelength (nm)  FIGURE 10. OPTICAL SPECTRA OF CTAB AND PEG 10000 COATED GOLD NANORODS.  Nanorods were kept in lower levels of CTAB after synthesis, as after two centrifugation/redispersion steps enough CTAB is still adsorbed on the surface to keep the particles stable. The approximate concentration of CTAB can be estimated to be 50µM based on the work done by Rostro-Kohanloo et al. [46]. An additional centrifugation step further removes CTAB, leading to aggregation and an extinction that goes close to baseline. Stabilization without CTAB was achieved by coating with PEG. The optical spectra of CTAB and PEG coated gold nanorods is shown in Figure 10. The concentration of CTAB stabilized nanorods was calculated to be approximately 1.1 x 1011particles/mL based on the longitudinal absorption maxima (Figure 10). Coating with PEG 10000 results in a red shift in the absorption maxima by ~40nm, therefore the spectra of nanorods can easily identify whether they were coated. The zeta potential was also an indication of a complete coating. CTAB nanorods had a large positive potential and PEG coated nanorods had a potential close to 0mV. This proves CTAB was removed, because PEG creates a neutral surface by electrostatic shielding [23]. Figure 11 shows a TEM of PEG 10000 coated nanorods, the light regions around the particles is PEG. From this image, the coating was approximately 10nm thick. Nanorods coated with PEG 2000, 10000 and 20000 did not exhibit any difference in 21  stability in solution. However, work by Niidome et al. showed nanorods had the highest stability in blood and best biodistribution when coated with PEG 5000 and 10000 [23].  FIGURE 11. TEM IMAGE OF PEG 10000 COATED GOLD NANORODS.  Synthesis of nanorods was simple, had a short procedure and produced particles with excellent optical properties. However, there is a great percentage of material wasted, as only ~15% of gold is predicted to form rods [43]. Also, a potential issue is photothermal reshaping of particles after heat treatment [17]. The work by Takahashi et al. revealed that after laser exposure, the nanorods indicated in their absorption spectra some reshaping into spherical nanoparticles [40]. Further study may be required to determine if this will affect the overall heating efficacy of nanorods in photothermal therapy. It is not well known whether CTAB concentrations needed to maintain particle stability are cytotoxic. If gold nanorods are produced on a large scale and CTAB cannot be present even after replacement with another stabilizing agent (e.g. PEG), complete detoxification may be difficult as CTAB can be hard to remove. However, surfactant exchange and dialysis techniques do offer some promise [17]. 2.5 Synthesis of Gold Core-Corona Nanoparticles 2.5.1 Introduction The development of novel types of nanoparticles with new properties is an active area of research. An example is the core-shell-corona nanoparticle, which consists of three layers, in Figure 12. Traditionally, this form of particle refers to composite gold-polymer micelles on a polystyrene core [47]. This layer-by-layer assembly creates core/shell architectures by 22  adsorbing oppositely charged polymers (n2) to a core particle (n1) and then adsorbing the charged gold colloids to make up the shell (n3) [26]. Preston and Signorell have developed a derivative of this particle type with absorption in the NIR and a total size under 100nm [38]. This core-shell-corona consists of a gold core (D = 18nm), coated with alternating layers of polymers (2nm total thickness) and finally an outer gold corona layer with thicknesses of 15 to 40nm. The corona contains rods of 8nm in diameter branching away from the surface, giving the particle surface a rough, brush-like appearance. The NIR absorption of these particles was determined to originate from complex plasmon coupling between rods in the corona and coupling between the rods and the inner gold core [38]. Tuning of the overall plasmon resonance is done by adjusting the corona thickness or length of rods.  FIGURE 12. ILLUSTRATION OF A CORESHELL-CORONA PARTICLE. ADAPTED FROM: [38].  The synthesis of core-shell-corona particles consists of simple steps that consist of long reaction times, as the total synthesis of the particles spans over seven days as a result of the polymer layering [38]. One of the desired features is the NIR absorption generated by the corona. Therefore, if this sole feature is isolated, the rod growth becomes the time limiting factor in the synthesis. Rods require up to two days to grow of sufficient length and density, as the plasmons couple due to their close proximity on the surface [38]. Parts of the coreshell-corona synthesis resemble the steps to generate nanoshells. The main difference is the use of a polymer as a scaffold to absorb gold seeds. The synthesis may be shortened by using a silica core as n1 and n2 in Figure 12, removing the five step layering of polymers, and 23  generating a core-corona particle instead. However, the only disadvantage of this shortened synthesis is the loss in plasmon contribution from the coupling of the corona and the core, as the gold core is replaced with silica. If the core-corona particles can be generated with similar properties to the core-shell-corona particles, the particles could be more feasible for an increase in synthesis scale and therapeutic applications. 2.5.2 Preparation of Gold Core-Corona Nanoparticles A simplified procedure to generate the brush-like features outlined in the method by Preston and Signorell [38] was performed. Instead of a gold core with polymer layers, a silica core was used to grow the corona. The procedure follows the same steps as outlined in Section 2.3.2, except for the shell growth stage.  Growth of Brush-like Features The reduction process was altered to promote the growth of the rods rather than a shell. A more concentrated gold solution was used to grow the corona to promote the growth of rods. This was done by performing fewer dilutions to the stock gold solution. The seeded silica cores (200µL) were added and the solution mixed before formaldehyde (30%, 20µL) was added. Reactions were mixed for 1 to 2 days. The UV-vis absorption was measured and TEM confirmed the brush-like features. Particles that exhibited the correct properties were centrifuged (14000rpm) for 30 minutes and redispersed in sterile water. The concentration of particles can be calculated by the same method as the nanoshells, because the same silica core concentration was used. 2.5.3 Results The alternative procedure to core-corona nanoparticles was far less laborious than the synthesis route to generate the core-shell-corona. The same brush-like features were obtained for the core-corona particles (Figure 13). Rods were randomly located on the surface of the silica sphere in the same way as the core-shell-corona. Therefore, growth was not changed by the use of a different scaffold. Sizing by TEM shows the particles to be 78 ± 11nm. Therefore, the corona thickness was about 25nm, which is consistent with particles generated by Preston and Signorell that absorb in the NIR [38]. Typical particle concentrations were similar to those of 40nm nanoshells, at 2.2 x 1011particles/mL. 24  FIGURE 13. TEM IMAGE OF GOLD CORE-CORONA NANOPARTICLES.  The core-corona particles also have a similar extinction spectrum (Figure 14) as the core-shell-corona particles produced by Preston and Signorell, proving the plasmon coupling phenomenon of the corona is consistent with that of the core-shell-corona [38]. The mechanism for the rod growth has not been described, but was attributed to the increase in concentration of gold and extended reaction time. As the seeds grow in size, growth seems to be favored on the ends. Formaldehyde may also have a role in mediating nanoparticle growth by preferential reduction. However, this was not proven and will need to be investigated further. These particles had the same PEG coating method as nanoshells. It was expected that the coating may not be complete or uniform, as a result of the extremely rough surface created by the rods causing increased steric hindrance between PEG chains. Here a shorter PEG chain length would be advantageous.  25  FIGURE 14. OPTICAL SPECTRA OF GOLD CORE-CORONA NANOPARTICLES.  These particles do exhibit interesting and unique properties, with a strong absorption in the NIR. Although the original synthesis was shortened, the procedural method to generate rods is not well developed or optimized. Therefore, the route may still not be appropriate for a scale-up unless further work is performed. The properties of this particle may have some similarities to gold nanoshells. However, the optical and thermal robustness of the particle is not known. The stability of the corona must be studied to see if this structural feature will suffer from collapse upon laser irradiation or heating. There is no documented data on the thermal efficiency of such a nanostructure and if cellular uptake is affected by a rough surface. 2.6 Synthesis of Hollow Gold Nanoshells 2.6.1 Introduction Hollow gold nanoshells are nanoshells that have aqueous interiors (no solid core) [26]. They can be synthesized by reducing gold by a galvanic replacement reaction onto solid templates, such as silver nanoparticles. The size, shape and void space are determined by the selected template and the shell thickness depends on the reagent ratios. The SPR band may be tuned by adjusting the overall diameter and shell thickness, similar to the gold nanoshells with a silica core (Section 2.3). An increasing particle size with a constant shell thickness, 26  results in a red shift in the absorption, where as increasing shell thickness at a constant particle size results in a blue shift [20]. In template galvanic replacement reactions the core particle is oxidized by the metal salt that is reduced to form the shell. This occurs because the shell metal has a greater standard reduction potential than the template metal [48]. The redox reaction using a silver template to form a gold particle is as follows, 3Ag(s) + HAuCl4 (aq)  Au(s) + 3AgCl(aq) + HCl(aq) The gold is mainly confined to the template surface and first nucleates very small particles that evolve into a thin shell over the silver template. The shell has an incomplete structure as the layer forms, because both gold and silver are continuously diffusing across until the template is completely consumed (Figure 15). The gold shell then reconstructs producing a highly crystalline structure, forming a smooth and seamless coating [48].  FIGURE 15. SCHEMATIC OF HOLLOW GOLD NANOSHELL SYNTHESIS. ADAPTED FROM: [49].  The galvanic replacement is a short synthesis method and deemed to be highly scalable, as it uses minimal amounts of toxic reagents and has minimal steps [26]. The greatest benefit of producing nanoshells by this method, versus those with a solid core, is the ability to construct very thin shells. Hollow gold nanoshells produced by Schwartzberg et al. yielded shells as thin as 4nm for particles below 30nm [20]. This is beneficial as the synthesis can produce small nanoparticles that can be tuned to absorb in the NIR. 2.6.2 Preparation of Hollow Gold Nanoshells Synthesis of Silver Spheres Reduction of water soluble metal salts represents the simplest and most common bulk synthetic technique for metal nanoparticles. The metal nanoparticle growth process is 27  normally controlled by a stabilizing agent, which manipulates the size and shape. Various reduction and stabilizing agents can be used, however, dual role agents that can perform both functions are available. This is desirable in order to simplify the total synthesis. An example of a molecule that can serve a dual role, to form spherical silver nanoparticles, is citrate. The citrate reduction reaction used was based on the well known Turkevich method [50]. In a 100mL round bottom flask, 50mL water was heated in an oil bath. Silver nitrate (9mg) was added to the flask and the mixture was brought to a boil, before adding sodium citrate solution (1%, 1.0mL). The amount of citrate controls the size of the nanoparticles, as variation in citrate concentration changes the reduction rate and the nucleation-to-growth ratio [51]. The solution was refluxed and stirred for at least 30 minutes. Generally, the color of the solution changed from colorless to yellow, then turbid gray, indicating the reaction was complete. The particles were characterized by their UV-vis absorption and DLS. The concentration of the silver particles may be determined as described in Section 2.3.2.  Synthesis of Hollow Gold Nanoshells by the Galvanic Replacement Reaction The process used was a modified version of Au et al. procedure to produce in-situ polymer coated nanoparticles [52]. In a small round bottom flask, silver spheres (2.5mL) and PVP solution (0.1g/mL in water, 7.5mL) was added. The mixture was brought to a boil and HAuCl4 (0.2mM, 4.0mL) was added rapidly. Upon addition of gold the solution changes color to a blue/gray within seconds. The solution was refluxed for 5 to 10 minutes and purified by stepwise centrifugation. Visible precipitates, which were most likely silver byproducts, remain in solution and were removed first by centrifuging at a low centrifuge speed (2000rpm). The supernatant was collected leaving behind the pellet of by-products. This process was repeated as needed. The hollow gold nanoshells were centrifuged (14000rpm) and redispersed in sterile water. The UV-vis absorption was measured. TEM was used to confirm size and shape. Particle concentration may be determined based on the silver particle concentration (Section 2.3.2), assuming all templates are converted. 2.6.3 Results Generating hollow gold nanoshells by galvanic replacement is a quick method to produce particles that absorb in the NIR. The procedure chosen in this present study was a shortened method involving a few basic steps. However, there are several documented 28  methods using various templates and reaction conditions (e.g. temperature, addition method and concentration) [20]. Usually improvements in size distribution or morphology, to optimize the final product, will complicate and extend the procedure significantly. The method choice here utilized simple reaction steps and minimized purification steps.  FIGURE 16. TEM IMAGE OF HOLLOW GOLD NANOSHELLS.  The silver templates used were spheres with an average diameter of 60nm that had an absorbance maximum at 428nm. Hollow gold nanoshells generated from these templates had an exterior diameter of approximately 85 ± 17nm with an expected interior diameter around 60nm. The shell thickness was not well defined for all particles, however can be estimated from the TEM to be 10-15nm (Figure 16). It is evident that the nanoshells were hollow, as the centers look significantly lighter in the TEM image. This proves the silver template particle was removed. The particle concentration for a typical synthesis of hollow gold nanoshells was 3.4 x 108particles/mL.  29  FIGURE 17. OPTICAL SPECTRA OF HOLLOW GOLD NANOSHELLS.  Residual silver spheres were not present in the final product which was shown by the absence of a contribution at 428nm in Figure 17. Also, there was no secondary nucleation of pure gold particles (520nm). The PEG coating of the hollow gold nanoshells was done with PEG 20000 and the absorption maxima exhibited a red-shift similar to that of the nanorods. The broad SPR band is likely the result of the combined resonances of inhomogeneous particles. Depending on the synthesis, hollow gold nanoshells can be considered to be polydisperse in both shape and size [26]. Prevo et al. irradiated samples at select wavelengths and removed portions (hole-burning) of the absorption peak as particles collapsed into solid spheres [26]. The silver templates used in this work had a large size distribution, as particles < 10nm were also present. However, the reaction with gold results in the disappearance of these very small particles, as they were not present in the TEM of the final product (Figure 16). These particles were presumed to be lost as silver by-products and removed in the stepwise centrifugation step, or potentially adsorbed on the walls of the shells. The optical properties of hollow nanoshells are very similar to that of solid core nanoshells, as they both depend on core-to-shell manipulation. However, the route to a hollow core has the potential for thinner shells to be made for smaller particle sizes [20]. The synthesis was simple and generates gold nanoparticles with appropriate optical properties that can be used in photothermal applications. But, there are issues that need to be addressed 30  before scale-up is considered. Removal of residual silver by-products was a disadvantage, as it resulted in a loss of hollow gold nanoshells. Although silver by-products may be removed by purification techniques, it has also been documented that complete removal of silver is not possible. Silver is still measured to be present, most likely incorporated in the hollow gold shell [26]. Work by Au et al. estimated that particles produced by the galvanic method contained up to 37% silver [52]. Although silver does exhibit antimicrobial properties [26] it is unknown if this will be beneficial or harmful for in vivo use. Another issue that may affect the phototherapeutic use of these nanoparticles is that hollow structures have been proven to not be thermally robust. Short periods of laser irradiation collapses the nanoshells into smaller solid nanoparticles resulting in an absorbance shift out of the NIR, limiting the nanoparticles active use in thermal therapy [26]. Whether these issues will produce problems downstream for medical applications will need to be further studied. 2.7 Discussion Quite a few approaches to generating gold nanoparticles exist, with wet chemical methods often being the preferred choice because of simplicity. The aim in this chapter was to prepare gold nanoparticles that were appropriate for photothermal therapy of prostate cancer. Particles under 100nm in size that absorbed in the NIR (800nm) were the main criteria. The quality of the particle was evaluated in terms of size, shape, presence of impurities/toxic reagents and stability with emphasis on finding a scalable route for practical applications. The length of the PEG chain did not significantly affect the stability of the particles overall. All types and sizes of gold particles were coated easily, as a result of the strong covalent bond formed between gold and sulfur. Although, the mechanisms governing particle growth for many gold nanoparticles are not well understood [53] most synthesis techniques, for common particle types, are well developed and easily controlled. Many researchers try to develop a true one-pot synthesis, but removing steps may result in a lower quality product. Therefore, a balance between quality in properties and economics must be found. Determining the nanoparticle concentration can be done by several methods, which were briefly discussed in this chapter. However, there is not a standard method of determining accurate nanoparticle concentrations with an associated level of certainty. All calculated concentrations were assumed to be good approximations of the actual particle concentrations.  31  For work in later chapters the absorbance was used as a measurable component to keep consistency between experiments, as absorbance is equivalent to concentration [43]. Among the nanostructures investigated here, gold nanoshells [13] and nanorods [54] represent some of the most effective agents for NIR photothermal therapy. Nanoshells have proven optical and thermal robustness and nanorods have a strong, highly tunable absorption. Their syntheses have been demonstrated to be simple and straightforward producing the smallest nanoparticles of the four particle candidates examined. All of the particles were further assessed in Chapter 3 by measuring cellular uptake. However, the hollow gold nanoshells and gold corecorona nanoparticles were used far less as their synthesis and/or particle properties were less favorable than the gold nanoshells and gold nanorods. The hollow gold nanoshells were used for a preliminary in vivo study in Sections 5.2.4 and 5.3.3.  32  Chapter 3. Cellular Uptake Studies of Gold Nanoparticles 3.1 Introduction Gold nanoparticles may be anchored to the outer membrane of cells or internalized, depending on the functionalization of the gold surface. In this work, uptake is referred to as internalization of the nanoparticles by the cells. Bare and PEG coated particles are taken up nonspecifically, as they are not functionalized to target the cell membrane. For photothermal therapy, the amount of gold nanoparticles taken up by the cells will determine the effectiveness of the treatment. Therefore, it is important to measure accumulation. If the particles are internalized, rather than specifically attached to the surface, the chances of cell necrosis are higher [19] as there is collective heating of the particles [18] within the cell. In Chapter 2, gold nanoparticles with the appropriate properties were synthesized to optimize cellular uptake. This chapter evaluates the uptake of these particles. All eukaryotic cells have a membrane system that allows the uptake of molecules and particles from extracellular fluid [55]. The mechanism that describes cellular internalization of gold nanoparticles is endocytosis. Although there are several different types of endocytosis, the general steps are interaction of the particle with the cell membrane, enclosure by a small portion of the membrane, invagination inwards, formation of a vesicle and lastly release into the cell interior (Figure 18) [21]. The reverse pathway is known as exocytosis.  33  FIGURE 18. GENERAL SCHEMATIC FOR ENDOCYTOSIS OF GOLD NANOPARTICLES IN A CELL. SOURCE: GENERATED USING CHEMBIODRAW ULTRA.  There have been several studies on elucidating the various pathways of endocytosis as it is mediated by several factors. Two main factors tend to be size and cytotoxicity of the material. The optimal particle size for uptake is determined by the cell surface available for vesicle formation and time for complete enclosure of the particle by the membrane [56]. Research by Chithrani and Chan determined that a gold nanoparticle size of 50nm had the fastest enclosure by the membrane, leading to a higher accumulation in the cell [56]. They also observed bigger particles ( > 50nm) were enclosed much slower and smaller particles required clustering before uptake occurred. Larger particles ( > 100nm) may also interrupt some cellular functions [20] and smaller particles ( < 20nm) may be interpreted as a toxin if not clustered [21]. This could result in the activation of defense mechanisms that could prevent uptake. Cellular toxicity is avoided as it is generally desirable to keep the cells healthy. If the material is toxic, cancerous and noncancerous cells may be injured or destroyed upon exposure. This makes the material nonviable for medical use in humans. One of the many benefits of gold is that it is inert and has been proven to be biocompatible and non-cytotoxic. There are many other factors that can affect uptake, such as particle shape, surface functionalization, concentration of particles and incubation time. Particle shape may affect the interaction of the nanoparticle with the cell membrane. Functionalization, for specific targeting 34  of the cell membrane, will determine whether the particle will be anchored to the surface, effect the mechanism of endocytosis and/or the surface charge of the particle. Most cell membranes are polarized such that the inside is negatively charged, creating a potential. Therefore, anionic structures are presumed to bind less efficiently to the cell membrane as compared to neutral or cationic structures, as a result of electrostatic repulsion [19, 21]. Concentration and incubation time can be used to tune the conditions to maximize internalization for given nanoparticle properties. The prostate cancer models chosen for this present study were, PC3 and LNCaP. These cancer cell lines closely reflect disease conditions, as they can represent early and late stages of prostate cancer [57]. PC3 is an androgen-independent cell line and LNCaP is androgendependent [58]. Both are attachment cells that grow readily and are commonly used as experimental systems for in vitro and in vivo work of prostate cancer. For example, PC3 exhibits good tumor growth for subcutaneous experiments on mice [59, 60] and LNCaP is a close representation of a human model as it is hormone dependent. The light scattering properties of gold nanoparticles can be used to image uptake in cells. In order to determine the success and degree of uptake, the cells were imaged by light scattering microscopy. This method offers the simplest method for detecting the internalization of gold. Although the extinction of small gold nanoparticles is dominated by light absorption, as scattering decreases with decreasing particle size, the amount of scattering should be sufficient to detect the particles used in this study. Particles as small as 20nm can be detected by light scattering [61]. This method is a qualitative observation of cellular uptake. However, it offers the ability to easily confirm the internalization of the gold particles. 3.2 Materials and Methods 3.2.1 Materials PC3 and LNCaP (human prostate cancer cells) were obtained from Dr. Martin Gleave, Prostate Center, VGH, BC, Canada. Dulbecco's modified eagle medium (DMEM), RPMI 1640 medium, fetal bovine serum (FBS), penicillin-streptomycin, phosphate-buffered saline (PBS, pH 7.4), trypsin (0.25%) and trypan blue stain (0.4%) were obtained from Invitrogen. Paraformaldehyde (95%), sodium hydroxide (NaOH, 99.8%), hydrochloric acid (HCl, 36.538%) and dimethylsulfoxide (DMSO, 95%) were obtained from Fisher Scientific. Thiazolyl blue tetrasolium bromide (MTT, 98%) was obtained from Alfa Aesar and used to prepare the 35  MTT reagent (2.5mg/mL in 1x PBS). All chemicals were used as received. Milli-Q water (18.2 MΩ.cm @ 25 °C) was used in all the experiments. 3.2.2 Imaging of Gold Nanoparticle Cellular Uptake by Light scattering Cell Preparation PC3 were cultured in DMEM and LNCaP in RPMI with 10% FBS and 1% penicillinstreptomycin at 37C under 5% CO2 to a confluence of 70 to 90%. The cells were detached by trypsin and counted using a hemocytometer and trypan blue staining. Cells were plated by adding approximately 1.5 x 105 cells per well in a 6 well tissue culture plate containing 18mm glass cover slips. Cells were grown for 1 day. Gold nanoparticles were added and incubated for 4 or 24 hours at 37C under 5% CO2. Cells were incubated with nanoparticle solutions that had similar absorbance values. Table 3 shows the typical incubation concentration ranges for PEG coated and bare particles. Images of live cells were taken using an Olympus 1X70 microscope with a 3CCD MT1 camera (Dage-MT1 Inc.) to document cell health. After incubation the cells were fixed for 10 minutes in paraformaldehyde (2%, 1mL). The cover slips coated with a layer of cells, were washed in PBS (1x, 1mL) twice and mounted in 50% glycerol in 1x PBS on a microscope slide. TABLE 3. TYPICAL INCUBATION CONCENTRATIONS OF GOLD NANOPARTICLES FOR CELLULAR UPTAKE STUDIES.  Nanoparticle Type 40nm Gold Nanoshell* 120nm Gold Nanoshell* Auroshell®** Gold Nanorod*** Hollow Gold Nanoshell* Gold Core-Corona*  Concentration Range (particles/mL) 5 x 109 to 7 x 1010 4 x 106 to 2 x 107 5 x 107 to 1 x 108 3 x 109 to 1 x 1010 2 x 106 to 1 x 107 5 x 109 to 7 x 1010  * Concentration based on Section 2.3.2 ** Concentration based on manufacturers values ***Concentrations based on Section 2.4.2  Description of set-up Light scattering microscopy was used to image the gold particles using a previously developed set-up [62]. In summary, images were taken by using a modified Olympus FV300 laser-scanning microscope with a laser centered at 532, 580 or 800nm at a power typically less than 0.5mW. A 60X objective lens was used to collect the scattered light from the 36  samples and detection was by a photomultiplier tube (PMT). Two (2D) and three dimensional (3D) images of the cells were taken to observe uptake. 3D images were obtained by capturing a stack of 2D (x and y) images along the z-axis to determine if the particles were internalized by the cells.  Measurement Procedure Control cells were measured first to establish a background level of scattering. One drop of water was placed on the objective lens and the slide was placed cover slip down. The laser was applied after the cells were put in focus. A transmission image was captured to verify cell locations. The cell scattering was observed and adjustments were made so the silhouette of the cells was visible and matched that of the transmission image. Samples containing gold were then measured and the images captured. The size scale of unmagnified screen captures was 235.7 x 235.7µm. 3.2.3 Nanoparticle Toxicity Test This experiment was performed using 40nm gold nanoshells synthesized in Chapter 2 and Auroshell® particles obtained from Nanospectra Biosciences Inc.. This procedure requires a period of 3 Days. PC3 and LNCaP cells were cultured as previously described. Cells were plated by adding 10,000 cells per well, to a total volume of 200µL, in a 96 well plate and the cells were incubated overnight (Day 1). Enough wells were plated to accommodate for triplicate analysis of control samples and samples with gold. Gold nanoparticles were added at the concentrations in Table 4, for bare and PEG coated particles, and incubated for 24 hours (Day 2). The cell viability was measured by the MTT assay (Day 3). The MTT assay is a quantitative assay that involves the metabolic reduction of yellow tetrazolium salt (MTT) in active cells to form water insoluble purple colored formazan crystals. MTT reagent (2.5mg/mL, 50µL) was added and incubated at 37°C for 3 hours. The supernatant was removed by vacuum aspirator by 27 gauge needle. The formazan crystals were dissolved in DMSO (150µL) and the optical density at 595nm was measured. Cell Viability (%) = (AT - AB)/(AC - AB) x 100%  (Eq. 6)  The cell viability was calculated using Eq. 6 where AT is the absorbance of the treated cells with gold, AB is the absorbance of the blank and AC is the absorbance of the control. The data was subject to statistical analysis using analysis of variance (ANOVA) with a p 37  value of 0.05. ANOVA and a Bonferroni's multiple comparison tests were performed using Graphpad Prism 4. When two or more samples were compared, if p < 0.05 the difference between the results was considered to be significant, if p > 0.05 the difference was not significant. TABLE 4. GOLD NANOPARTICLE INCUBATION CONCENTRATIONS FOR TOXICITY ASSAY.  Nanoparticle Type 40nm Gold Nanoshells* Auroshells®**  Incubation Concentrations (particles/mL) 1 2 3 1 x 1010  2 x 1010  4 x 1010  6 x 107  1 x 108  2 x 108  * Concentration based on Section 2.3.2 ** Concentration based on manufacturers values  3.3 Results 3.3.1 Interpretation of Images When cellular uptake of gold nanoparticles is determined by light scattering, the key is to observe the level of illumination of the particles against the cell background. Mammalian cells exhibit minimal scattering, therefore any scattering can be assumed to originate entirely from gold nanoparticles. The transmission image in Figure 19 of control PC3 cells clearly identifies cell locations. In the corresponding scattering image, cells were less visible.  FIGURE 19. TRANSMISSION (LEFT) AND SCATTERING (RIGHT) IMAGES OF CONTROL PC3 CELLS.  38  PC3 and LNCaP are attachment cell lines which allow them to be easily imaged by light scattering. Both cell types spread and extend in various directions on the cover slip surfaces. Most of the images displayed in this chapter have a cell shape that is easily identified by the gold nanoparticles within the cell, if there is sufficient gold nanoparticle uptake.  FIGURE 20. TRANSMISSION (LEFT) AND SCATTERING (RIGHT) IMAGES OF LNCAP INCUBATED WITH 110NM GOLD NANOSHELLS.  Figure 20 demonstrates the scattering of gold in a group of LNCaP cells and shows the clear location of the cells (right). Cell location can also be confirmed by the transmission image of the same screen capture (left). The uptake of gold was established by the degree of illumination of the cell shape. The border and interior of the cells were well defined by the scattering in Figure 20, indicating good uptake. If the cellular uptake was low (no scattering), the silhouette of the cell shape was seen (Figure 19, right).  39  FIGURE 21. CELLULAR UPTAKE COMPARISON BETWEEN LNCAP (LEFT) AND PC3 (RIGHT) WITH 40NM GOLD NANOSHELLS.  In most experiments, PC3 demonstrated more cellular uptake than LNCaP. Both cell lines exhibited the same cellular uptake trends for all particles, however, images of PC3 seemed to have consistently more scattering and distribution throughout the cell. Figure 21 compares the incubation of both cell lines with the same incubation concentration of 40nm gold nanoshells (2 x 1010particles/mL). Upon examination of single cells in the images, PC3 cells show more scattering. These differences in cellular uptake may be attributed to different cellular properties between the two cell lines. However, the amount of difference was not quantifiable based on this method. It can be assumed in this work, that PC3 and LNCaP may have different levels of cellular uptake, but overall exhibit similar uptake trends. 3.3.2 Uptake of Gold Nanoshells The overall cellular uptake of gold nanoshells was moderate to good for all sizes. However the smallest nanoshell (40nm) for the given experimental conditions, exhibited the best internalization by the observed light scattering. Figure 22 shows the comparison of 40nm nanoshells to the 150nm Auroshell® particles. Cells were incubated with nanoparticle solutions with similar absorbance values. The incubation concentrations were determined to be 1 x 108particles/mL for Auroshells® and 2 x 1010particles/mL for 40nm gold nanoshells in Figure 22. However, concentration calculations by ICP-MS estimate that these concentrations were closer in value at 2.2 x 1011 and 7.8 x 1012particles/mL, respectively.  40  FIGURE 22. CELLULAR UPTAKE COMPARISON FOR 40NM GOLD NANOSHELLS (LEFT) AND AUROSHELL® PARTICLES (RIGHT) IN PC3 CELLS.  Small nanoshells had the most consistent observed uptake with accumulation across a greater portion of the cell. This result may be attributed to the small size and also due to differences in concentration, as actual particle concentrations were not known accurately. Theoretically, if the cells were incubated with identical particle concentrations, measurement by light scattering would still not be an accurate assessment of internalization, as the scattering efficiency changes with particle size. The larger the size, the particles are expected to scatter more light. Therefore increased scattering of the larger Auroshells® still hinders direct comparison. However, the higher scattering of Auroshells® should act to offset a prospectively larger particle concentration of smaller nanoshells. If the concentrations were close in value, the lower scattering in Figure 22 for the Auroshells® is a strong indication that the uptake of 40nm nanoshells is better. Several studies have confirmed smaller particles do have a higher accumulation in cells [19, 56, 63], which can support this claim. The 3D images in Figure 23 show the distribution of gold nanoshells in a single cell. The image shows the cell at a certain z position (top left) with cross sections along the x and y axis (right and bottom panels). The glowing bands present in the panels are due to the reflection of the microscope slide and cover slip. When the cell is examined at several different x, y and z positions, internalization can be fully observed. The 40nm nanoshells had the best distribution throughout the cell in comparison to the Auroshells®.  41  FIGURE 23. 3D SCATTERING IMAGES OF A SINGLE PC3 CELL INCUBATED WITH AUROSHELL® PARTICLES (LEFT) AND 40NM GOLD NANOSHELLS (RIGHT). ARBITRARY MAGNIFICATION.  When comparing bare (uncoated) and PEG coated particles, there was less observed internalization for PEG coated particles for all nanoshell sizes. For equivalent particle incubations of 5 x 107particles/mL of bare and PEG coated Auroshells®, the uptake was visibly different (Figure 24). This result was consistent with the fact that PEG coated particles result in less interaction with the cell membrane leading to a lower uptake [63, 64]. This property is what gives PEG coated nanoparticles a “stealth property” as the coating reduces detection by biological systems, which includes cell membranes [16, 23].  FIGURE 24. CELLULAR UPTAKE COMPARISON OF BARE (LEFT) AND PEG COATED (RIGHT) AUROSHELL® PARTICLES IN PC3.  42  Bare gold nanoshells synthesized in Chapter 2 and the nanoshells obtained from Nanospectra Biosciences Inc. were both assumed to be stabilized by anions, most likely chloride ions, giving the nanoparticles an overall negative surface charge (zeta potential). However, most cells have a membrane potential [21] which would lead to repulsion of the particles and less uptake. Chithrani et al. reasoned that the uptake of negatively charged particles was facilitated by nonspecific adsorption of serum proteins (FBS) to the gold surface, inducing internalization by receptor-mediated endocytosis [19]. This could explain part of the overall uptake, as endocytosis of the bare particles does not seem to be adversely affected by the surface charge at these particle sizes. Some basic concentration studies were performed in the concentration ranges outlined in Table 3. However, in this concentration range uptake was not significantly different in the light scattering images. If the concentration was raised much higher, gold nanoshells exhibited higher aggregation outside the cell and less efficient uptake. The aggregates settled on the cover slips, resulting in a high scattering background in images. Overall, nanoshells exhibited good uptake into the cells with no visible toxicity at the selected incubation concentrations. The smaller nanoshells can be presumed to have a higher uptake, as they have the best visible distribution. 3.3.3 Uptake of Gold Nanorods Cells that were incubated with CTAB stabilized nanorods at an incubation concentration of 3 x 109particles/mL exhibited a toxic response. The cells were visibly unhealthy after 4 hours and by 24 hours demonstrated cell death. Figure 25 illustrates the effect of CTAB on the cells. Healthy LNCaP cells were attached and spread across the surface (left) and unhealthy/dead cells were no longer attached and formed clusters (right). A similar response was observed in PC3 cells.  43  FIGURE 25. MICROSCOPE IMAGES OF LNCAP WITH (RIGHT) AND WITHOUT (LEFT) CTAB COATED GOLD NANORODS AFTER A 24 HOUR INCUBATION. MAGNIFICATION 100X.  Samples that were incubated with PEG coated nanorods remained healthy for the entire duration of the incubation. Therefore, it can be assumed that CTAB does cause a cytotoxic response, in PC3 and LNCaP cells, at concentrations needed to keep the nanorods disperse in solution. Because of this result only PEG stabilized nanorods were used for experiments.  FIGURE 26. SCATTERING IMAGES OF A PC3 CELLS: WITH (RIGHT) AND WITHOUT GOLD NANORODS (LEFT). ARBITRARY MAGNIFICATION.  In comparison to the light scattering images generated by the nanoshells the nanorods demonstrated far less scattering in both cell lines (Figure 26). Images collected completely lacked or exhibited very minimal scattering. If there was scattering present it was contained in a few cells upon scanning of the microscope slide. 3D scans of single cells showed very poor internalization. The results demonstrate a low uptake of nanorods or that the scattering was not detectable by the current set-up. Work by Chithrani et al. provided some general 44  guidelines in controlling the nanoparticle accumulation in cells for various particles of differing size and shape [19, 56]. Their work suggested that the optimal incubation time for gold nanorods is approximately 4 hours, as exocytosis may act to remove nanorods from the cell interior. However, when cells incubated at 4 and 24 hours were compared, there was no difference in observed scattering. Increases in concentration did not improve uptake. In an attempt to improve scattering of the nanorods, the sample was imaged at 800nm rather than a laser centered at 532 or 580nm. Scattering is enhanced at the SPR frequency as there is an electric field enhancement due to the collective oscillations of the conduction electrons (Chapter 2). Images generated by the 800nm laser (Figure 27) gave similar scattering results to previous images. However, the overall intensity of the image was greatly reduced, as the silhouette of the cells was less defined.  FIGURE 27. SCATTERING IMAGE OF LNCAP CELLS WITH PEG 10000 COATED GOLD NANORODS. IMAGED AT 800NM.  There is some question to if nanorods can be imaged by light scattering. Some studies show nanorods can be detected by light scattering [17][61] while others have demonstrated they do not scatter sufficiently to be visible [46]. Work by Rostro-Kohanloo et al. demonstrated 50nm x 15nm nanorods could be detected by two photon luminescence and atomic force microscopy but not by light scattering [46]. However, this is expected to be dependent on the configuration of the microscope and detector. Pure nanorods were 45  examined by the imaging set-up described in Section 3.2.2, to determine if some scattering was detectable. Scattering was not detected, which agrees with the results of RostroKohanloo et al. [46]. Uptake of nanorods could not be confirmed or refuted by the current method. Some studies have shown, by other analysis methods, that there is cellular uptake of nanorods in other cancer cell lines, such as SKBR3 [46] and Hela cells [19]. A few comparative studies in literature [19, 56, 63] show that spheres have a higher cellular uptake than rods. Chithrani et al. rationalized the difference by the shape of the nanoparticle [19, 56]. Rods have a larger contact area with the cell membrane and therefore less membrane is available for enclosure, leading to a lower uptake. Uptake may also be further hindered by the PEG coating. The low scattering may also be accredited to the nanorod dimensions as they may contribute to visibility issues, as the width dimension is ~20nm. Any scattering observed in Figure 26 may be nanorods. However, they may also represent the small fraction (5%) of non-rod shaped particles present in the incubating solution. 3.3.4 Uptake of Gold Core-Corona Nanoparticles Few uptake studies were done on gold core-corona nanoparticles as they had low to moderate uptake and the particles exhibited aggregation in cellular uptake experiments. Figure 28 demonstrates the visible aggregates, for a 2.9 x 1010particles/mL incubation, present in the transmission image (left) and the corresponding scattering image (right). Aggregates were present outside of the cell leading to a less efficient uptake. PEG coating did not improve the uptake for similar reasons discussed earlier in Section 3.3.2, but did improve particle stability leading to lower aggregation.  46  FIGURE 28. TRANSMISSION (LEFT) AND SCATTERING (RIGHT) IMAGES OF LNCAP INCUBATED WITH GOLD CORE-CORONA NANOPARTICLES.  Some preliminary uptake studies done with core-shell-corona particles produced by the procedure outlined by Preston and Signorell [38], showed very poor uptake in PC3 and no uptake in LNCaP. Lower cellular uptake may be attributed to less favorable interactions with the cell membrane possibly because of the extremely rough surface of the particle or particle aggregation. 3.3.5 Uptake of Hollow Gold Nanoshells The cellular uptake of hollow gold nanoshells was moderate. Figure 29 shows the scattering image for PC3 with the particles (~6.2 x 106particles/mL). Increases in concentration did not appear to affect the uptake significantly in the ranges outlined in Table 3. Higher concentrations lead to more aggregation outside the cell. The difference in uptake of the hollow nanoshells versus the solid core nanoshells may be due to several factors, such as size, concentration and surface properties. Bare hollow gold nanoshells were stabilized with PVP, which may lead to a different interaction with the cell membrane or interference with adsorption of serum proteins (Section 3.3.2). PEG coating did not improve the cellular uptake.  47  FIGURE 29. CELLULAR UPTAKE OF PC3 INCUBATED WITH HOLLOW GOLD NANOSHELLS.  3.3.6 Nanoparticle Toxicity Test A representative toxicity assay was completed for 40nm gold nanoshells (NS40) and the Auroshell® particles (NSAS) to determine if there was any cytotoxicity to cells at the incubation concentrations. An arbitrary set of concentrations, within the ranges in Table 3, were chosen to determine if the cell viability was affected after a 24h incubation period.  48  FIGURE 30. CYTOTOXICITY PROFILES OF LNCAP (LEFT) AND PC3 (RIGHT) INCUBATED WITH BARE AND PEG COATED 40NM GOLD NANOSHELLS (NS40) AND AUROSHELL® PARTICLES (NSAS). CONCENTRATIONS OUTLINED IN TABLE 4.  Analysis by ANOVA suggests that there was no significant difference (p > 0.05) in cell viability between the control samples and the samples incubated with the gold nanoparticles. The graphs in Figure 30 suggest there may be a slight decrease for samples incubated at the highest concentrations, but this was considered to be statistically insignificant. Therefore, at these incubation concentrations 40nm gold nanoshells and Auroshells® can be considered to be non-toxic. 3.4 Discussion The light scattering properties of gold nanoparticles can be used to image cancer cells and detect the particle uptake in vitro. Although, light scattering microscopy is not a quantitative technique, it can confirm successful uptake easily without the need for extra labeling steps (e.g. fluorophore) or laborious techniques, such as cell mapping with TEM. Verification of uptake 49  and an approximate assessment of the uptake were adequate measures for research purposes in this present work. There are several quantitative uptake studies available in literature, using various types and sizes of particles that can be used as guidelines for cellular uptake [19, 56, 63]. Cellular uptake, in PC3 and LNCaP cell lines, of some of these particle types have not been studied in literature. So confirmation and comparison of the level of uptake was important to assess, which particles were appropriate for further investigation as a photothermal agent. For these prostate cancer models, the cellular uptake of gold particles based on light scattering images is summarized in Table 5.  TABLE 5. SUMMARY OF GOLD NANOPARTICLE UPTAKE IN PROSTATE CANCER CELLS.  Nanoparticle Type 40nm Gold Nanoshell Auroshell® Gold Nanorod Hollow Gold Nanoshell Gold Core-Corona  Observations Good uptake Moderate to good uptake No uptake or uptake not detectable Moderate uptake Low to moderate uptake  In all cases variations in concentration did not improve the uptake significantly in the images. Higher concentrations, than those outlined in Table 3, often resulted in particle aggregation outside the cells. PEG coating increased nanoparticle stability, but decreased uptake. Maximizing the uptake into cells was important to study the photothermal effect of the nanoparticles on prostate cancer cells. Therefore, bare particles were more favorable for use in therapy studies. The gold nanoshells did seem to have the best uptake and distribution in the cells. The 40nm nanoshells were studied further and compared against Auroshells® in in vitro photothermal studies (Chapter 5).  50  Chapter 4. Antisense Oligonucleotide Gene Therapy of Heat Shock Protein 27 4.1 Introduction Photothermal therapy is a treatment modality that destroys cancer by heat. Exposure to temperatures above physiological levels can induce cellular death (hyperthermia). However, for the hyperthermic treatment of cancer, the thermotolerance of cancer cells can be a major problem [59]. Cells have many defensive mechanisms that protect the cell from environmental or physiological stresses. Heat shock proteins (HSPs) are the most ancient defense system in all living organisms and are detected in all cells, as they are essential to survival [65]. HSPs are molecular chaperones that facilitate the transport, folding and assembly of proteins. They are highly expressed in cancer cells and are associated with thermotolerant and cytoprotective functions [65]. The presence of HSP improves cell resistance and results in a higher temperature requirement to cause hyperthermia. Treatment efficiency may be improved if photothermal treatment is combined with strategic removal of HSPs by gene therapy. There are several types of HSPs, each having many documented functions [66]. They are generally characterized into families according to their molecular weight, examples are, HSP90, HSP70 and small HSP. HSPs are upregulated when cells are exposed to low or moderate heat shock (42C) [55] or any extreme change in the cell’s environment. An increase in expression can be detected after 3 hours [67], with a maximum expression occurring at 16 to 18 hours [66]. High levels of HSPs have been documented to be present up to 6 days after exposure to the initial stress [67]. Although these proteins are commonly known for their protective functions, HSPs are also essential in cell homeostasis and pathogenic processes [68, 69]. Therefore, certain types of HSPs may be present even under non-stressed conditions. HSP27, which is classified as a small HSP, has increased expression in a variety of cancers, including prostate cancer. It is important because it has been found to mediate several cytoprotective functions in cancer cells [69]. It is associated with drug resistance, thermotolerance [59], increased tumorigenicity and inhibition of apoptosis [69, 70]. Work by Cornford et al. has also suggested that the expression of HSP27 can be used as a diagnostic tool to predict the clinical outcome of patients with prostate cancer [68]. For the prostate cancer models used in this study, under physiological conditions HSP27 is expressed in PC3 cells [59] and is weakly expressed in LNCaP [68]. Overexpression of HSP27 in LNCaP also causes the cells to become androgen-independent [71]. 51  Hyperthermia is commonly defined as the application of heat to destroy tissue by causing irreversible cell damage [72]. There are not any universally accepted standards for the conditions of hyperthermia, such as temperature and treatment time. However, an example of a classification system for hyperthermic conditions is given by Stauffer and Goldberg in Table 6 [73]. TABLE 6. HYPERTHERMIA CLASSIFICATIONS [73].  Hyperthermia Classification Low Moderate High  Temperature Range (C) 40 to 41 42 to 45 > 50  Treatment Time 6 to 72 hours 15 to 60 minutes 4 to 6 minutes  The low and moderate temperature hyperthermia conditions cause numerous subtle changes in tissue physiology, such as increased blood perfusion and vascular permeability [73], and can also sensitize cells [74]. However, for these conditions heat treatment alone is not enough to completely destroy cancer cells. The risk of using non-lethal (low and moderate) hyperthermic conditions is that surviving cells can develop thermotolerance if the cancer is not completely destroyed. Cells typically die either by apoptosis or necrosis. Hyperthermia is an example of necrosis. Heat induced cell injury/death is proposed to be the loss of cell membrane integrity by perforation and/or blebbing [54], which is triggered by the denaturation and aggregation of proteins [75]. The thermal requirement to induce an efficient hyperthermic response can vary between different cell types [76]. However, temperatures greater than 65C are often required to completely ablate cancer cells and tumors [60, 77, 78]. These temperatures are considered to be lethal and are invasive, as they can damage surrounding healthy cells and tissue when put into clinical practice. In order to lower the temperature to noninvasive or less invasive levels (e.g. < 50C), the cellular defense system must be compromised to improve heat sensitivity. Blockage of HSP27 production has been chosen as it has been documented to have critical roles in prostate cancer [68]. The knockdown of HSP27 was done by inhibiting the transfer of genetic information from DNA to protein.  52  FIGURE 31. PROPOSED MECHANISM OF STRESS-INDUCED HSP SYNTHESIS IN CELLS. ADAPTED FROM: [66]. SOURCE: GENERATED USING CHEMBIODRAW ULTRA  Figure 31 illustrates the proposed stress-induced expression of HSPs. The transcription of HSP is controlled by heat-shock factor (HSF) which is bound to HSP [66]. When the cell is exposed to external stress, HSF separate from HSP, becomes phosphorylated and forms trimers. The HSF trimers enter the nucleus and bind to a segment of the HSP gene, leading to the transcription of HSP mRNA. Each mRNA molecule can be translated into protein molecules. Blockage of HSP can occur at transcription or translation. At translation, the use of antisense oligonucleotides (ASOs) can block a certain segment of mRNA preventing the synthesis of HSP27. The specificity of the antisense approach is based on Watson–Crick base-pairing interactions, which minimize toxic and non-specific effects [79]. Several studies have been done on the expression of HSPs in cancer cells [59, 66], the ASO-induced suppression of HSPs [75] and the effect of HSP suppression on sensitivity to treatments, such as chemotherapy [69, 80, 81] and radiation [82]. However, there are limited studies that examine the relationship between HSPs and hyperthermia. Work by Gabai et al. showed the downregulation of HSP72 increased the sensitivity of prostate cancer cell lines to hyperthermia [80]. Similar results were found by Rossi et al. with their work on HSP70 and HeLa cells [83]. The effect of HSP27 knockdown, in PC3 and LNCaP, was investigated in this present study to determine if it increased cell sensitivity to hyperthermia, thus enhancing the results of photothermal treatment by a “synergistic effect”. HSP27 was treated by HSP27 ASO, 53  in hopes to block synthesis of HSP27. Although ASO can be taken up by a wide variety of cell types, the exact mechanism is unknown, but is most likely by endocytosis if directly administered [81]. To ensure uptake, a liposomal carrier system was used to transfect the nucleic acids into the cells. Following the transfection, heat was applied and the cell viability was measured. 4.2 Materials and Methods 4.2.1 Materials Opti-MEM I Medium and Lipofectamine 2000 was obtained from Invitrogen. CellTiter 96® Aqueous Cell Proliferation Assay (MTS) was obtained from Promega. HSP27 ASO OGX-427 (3.5mM,) was obtained from Dr. Martin Gleave, Prostate Center, BC, Canada. All materials were used as received. See Section 3.2.1 for additional materials. 4.2.2 ASO Transfection and Heating Procedure The experiment was performed over a period of 3 to 4 days depending on test conditions.  Cell Preparation and Transfection PC3 and LNCaP were cultured as previously described in Section 2.3.2.. The experiment was prepared by incubating cells in 25cm2 culture flasks to a ~60% confluence or 2.5 x 105 cells were added to each well of a 6 well plate and incubated overnight (Day 1). Transfections were performed using Lipofectamine 2000 under serum-free conditions. Lipofectamine encapsulates nucleic acids in neutral aqueous centers and have a cationic outer layer allowing for optimal cellular internalization by endocytosis. The total amount of Lipofectamine added was 5µL per 250µL of the total volume of ASO-Lipofectamine complex made, as per the manufacturer’s instructions. Samples were also incubated with an equivalent amount of pure Lipofectamine to observe the effect on cell health. ASO addition was determined by the desired final incubation concentration which ranged from 50 to 500nM. A sample procedure for a 6 well plate with a final ASO incubation concentration of 100nM was as follows:  In a 1.5mL microfuge tube, Opti-MEM (1.2mL) and Lipofectamine (50µL) were mixed and incubated for 5 minutes (S1). In a separate tube, Opti-MEM (920µL) and 54  ASO (10µM, 80µL) were mixed (S2). For control samples, prepared Lipofectamine solution (S1, 250µL) was further diluted with Opti-MEM (250µL) (S3). The ASOLipofectamine complex (S4) was made by mixing the ASO (S2, 1mL) and Lipofectamine (S1, 1mL) solutions and incubating for 20 minutes. All media was removed from the wells and replaced with serum free media (1.5mL). The ASOLipofectamine complex (S4, 0.5mL) or the diluted pure Lipofectamine (S3, 0.5mL) was added to the well to make a total volume of 2mL.  After a 4 to 5 hour incubation period the medium was replaced with serum media or FBS was added at a final concentration of 10% (Day 2). Cells were incubated further for 8 to 15 hours.  Heating Procedure Cells (50,000 to 75,000) were transferred to 0.5mL microfuge tubes with a final volume of 200µL (Day 3). This was done by detaching cells with trypsin and counting as described previously (Section 3.2.2). Samples were done in duplicate or triplicate. Enough samples of each cell line were prepared for heating at select temperatures (42, 43, 44, 45, 48, 51, 54 and/or 60°C) with a 37°C as the reference, for set time intervals of t = 0, 15, 30, 60 and 150 minutes. Control samples for untreated and transfected cells, not exposed to any heat (t0), were used as the 100% viability reference (AC) for cell viability calculations (Section 3.2.3, Eq. 6). Heat sources used include incubators, water baths or heating blocks. Set temperatures for incubators and heating blocks did not vary more than ± 0.5°C, and for water baths ± 1°C. All samples were started at the same time and developed with MTT (Section 3.2.3) at each time point. MTT reagent (50µL) was added right after heat treatment and incubated at 37°C for 3 hours. Samples were centrifuged at 4000rpm for 2 minutes and the supernatant removed by pipette or vacuum aspirator. The formazan crystals were dissolved in DMSO (100 to150 µL), transferred to a 96 well plate and the optical density measured. Images of cells during incubation periods were taken using an Olympus 1X70 with a 3CCD MT1 camera (DageMT1 Inc.) to observe cell health.  55  Notes:   A preliminary experiment determined cell viability by the trypan blue dye exclusion assay. Three samples of the cell population of each temperature and time pair were counted using a hemocytometer. The cell viability was calculated per sample using Eq. 7. % Cell viability = # CA x 100 (Eq. 7) # CT CA is the numbers of cells counted that were alive and CT is the total number of cells that were alive or dead. The cell viability data (triplicate values) was analyzed by ANOVA as previously mentioned (Section 3.2.3).    A double transfection test was also performed by repeating the transfection step (4 Days) in order to increase transfection efficiency.    A control heating experiment was performed without transfection to determine the hyperthermic response of cells (Section 4.3.1).  4.2.3 ASO Concentration Test The effect of ASO concentration on cell viability was performed by the MTS assay, which is similar to the MTT assay, however the aspiration step and DMSO addition is removed. This procedure requires a period of 3 Days. PC3 and LNCaP cells were plated by adding 10,000 cells per well in a 96 well plate and incubated overnight (Day 1). The number of wells created accommodated for control samples and samples with varying concentrations of ASO (Day 2) done in triplicate. The transfection was performed by a similar procedure as in Section 4.2.2, however, different concentrations of ASO were prepared. Final ASO incubation concentrations were, 10, 30, 60, 100, 150, and 250nM. Final well volumes were 90µL. After a 4 to 5 hour incubation period the medium was replaced with serum media and incubated for 15 hours. MTS reagent (20µL) was added to wells, incubated for 3 hours and the optical density was measured directly at 450nm (Day 3). The cell viability was calculated as described in Section 3.2.3.  56  4.3 Results 4.3.1 Hyperthermia of LNCAP and PC3 A hyperthermic profile for LNCaP and PC3 is shown in Figure 32. Temperatures up to 54C were examined over a long time period to observe the effect of heat on cell viability. Thermal damage to cells was found to be dependent on temperature and time, with the most effect occurring at higher temperatures and longer incubation times.  FIGURE 32. HYPERTHERMIA PROFILES OF LNCAP (LEFT) AND PC3 (RIGHT) CELLS AT DIFFERENT TEMPERATURES AND HEATING TIMES. CELL VIABILITY BY MTT ASSAY.  LNCaP and PC3 exhibited similar responses to heat with a large difference in viability between 43 ± 0.5 and 48 ± 1C, as well as a dramatic decrease upon exposure to 54 ± 0.5C. The close results of 48 ± 1 and 51 ± 1C may be attributed to the use of different heat sources and any inconsistencies in measurement or maintenance of the temperature. A treatment temperature between 43 and 48C was of interest, as within this range there is a change between low and moderate heating conditions for LNCaP and PC3 (Table 6). For heating periods below 15 minutes, at these temperatures, 50 to 100% of cells were still viable. 4.3.2 ASO Transfection and Preliminary Heating Test A preliminary experiment performed on PC3 cells is shown in Figure 33. Cells were incubated with 500nM HSP27 ASO and heated at 37, 40, 43, 50 and 60C. A high concentration of ASO was initially selected to ensure a high transfection efficiency to observe if a dramatic treatment effect could be seen. Omitted results include: 40 ± 0.5C results as they show a close relationship to results at 37 ± 0.5C and samples incubated at 60 ± 0.5C as they reduced cell viability directly to zero. 57  FIGURE 33. PRELIMINARY TRANSFECTION EXPERIMENT ON PC3 USING 500NM ASO. UNTREATED (LEFT) AND TRANSFECTED CELLS (RIGHT). CELL VIABILITY BY TRYPAN BLUE DYE EXCLUSION ASSAY.  There was not a consistent difference in cell viability between untreated and transfected samples at all time points for each temperature. There was a slight decrease in viability observed at 37C upon transfection. Differences were observed at the following heating conditions: 50 ± 1C for 60 minutes and 43 ± 1C for 150 minutes. There was a 20 to 30% decrease (p < 0.05) in cell viability for transfected cells in reference to untreated in both cases. This suggests the knockdown of HSP27 may have reduced cell survival upon heating. However, the manual counting of the cells created uncertainty in the values. It was difficult assessing the number of dead cells accurately, as they were often found fragmented or in clumps. Therefore, the cell viability values may be unreliable. A new protocol was developed for ASO experiments as the population of dead cells may not be adequately quantified by trypan blue dye exclusion. The MTT assay was selected to determine cell viability. This assay simplified the analysis, as each cell type has a linear relationship between cell number and absorbance. It is less user dependent and may be more accurate in quantifying changes in cell numbers. 4.3.3 ASO Concentration Test In the preliminary test (Section 4.3.2) it was observed that PC3 had an initial response to HSP27 ASO, as cell viability at 37 ± 0.5°C was decreased upon treatment. A concentration test was performed to examine the effects of ASO on the cells. Increasing the HSP27 ASO concentration was found to decrease cell viability of PC3 and LNCaP (Figure 34).  58  FIGURE 34. ASO CONCENTRATION TEST ON PC3 (LEFT) AND LNCAP (RIGHT) AT 37°C. CELL VIABILITY BY MTS ASSAY.  All treatment concentrations were compared using a Bonferroni's pair wise comparison test. For PC3, each concentration had a significantly different treatment effect (p < 0.05). However, LNCaP only showed significant differences between 30 and the 100 and 250nM concentrations. The variance observed in the values for LNCaP may be the result of increased formation of cell clusters, leading to a less consistent number of cells per well. The looser surface attachment of LNCaP, compared to PC3, could also lead to losses in cell numbers as cells may be washed away during any additions or removals of the cell media. Results in Figure 34 were consistent with work by Kamada et al.. HSP27 ASO (OGX427) reduced the level of HSP27 and the corresponding cell viability in a dose-dependent manner in bladder cancer, with > 95% of measured HSP27 inhibited at a 50nM treatment level [69]. The similar trend observed for both PC3 and LNCaP cell viability, proved that the transfection of HSP27 ASO was successful. Knockdown of HSP27 did result in a noticeable change in cell condition (Figure 35) which can be explained by Rocchi et al. In their work they decreased HSP27 expression to undetectable levels in LNCaP and PC3 cells, resulting in a 2.4 to 4 fold increase in apoptotic cell death and 40–76% inhibition of cell growth [70].  59  FIGURE 35. MICROSCOPE IMAGES OF UNTREATED (LEFT) AND TRANSFECTED (RIGHT) PC3 CELLS. MAGNIFICATION 100X.  Representative images of untreated and transfected PC3 cells in Figure 35 show the treatment effect of 100nM ASO. Studies using pure Lipofectamine did not have an effect on cell viability, so the carrier system was considered to be non-toxic. 4.3.4 ASO Transfection and Heating Experiments Several experiments were performed using the MTT assay to determine the effect of treatment with ASO and heating on cell viability. In general, most experiments were performed using around 100nM HSP27 ASO to ensure an efficient transfection. Figure 36 shows the results of an experiment done on LNCaP. Transfected cells did exhibit an initial decrease in cell viability consistent with Figure 34. At 37 ± 0.5C the untreated (37C) and transfected (37C ASO) samples were significantly different (p < 0.05) with a consistent viability difference (gap) of up to 33% across all time points (Figure 36, top). Samples at 42 ± 1C had a similar result. Heating at 48 ± 1C showed an increased effect on cell viability over the 60 minute heating period, but a decreasing cell viability difference (gap) between untreated (48C) and transfected (48C ASO) samples. Viability differences between the 48C curves in Figure 36 (top) decreased from 30 to 0% over 60 minutes of heating. Only samples at 0 and 15 minutes for untreated and transfected samples were significantly different (p < 0.05). It is known that the knockdown of HSP27 has an initial effect on cell viability. Therefore, to determine the effect of heating, the effects should be isolated. For an incubation concentration of 100nM, the reduction in cell viability for LNCaP from Figure 34 should be ~40 to 50%. Transfected samples heated at 37 and 42C had a reduced cell viability of ~33% that was consistent for the entire 60 minutes of heat treatment. So for these temperatures, 60  intuitively, the decrease in cell viability can be assumed to be exclusively the result of HSP27 knockdown, which leads to apoptosis and inhibition of proliferation. However, this deduction does not work for data at 48C.  FIGURE 36. REPRESENTATIVE TRANSFECTION AND HEATING EXPERIMENT ON LNCAP WITH 100NM ASO. ASO TRANSFECTED CELL VIABILITIES CALCULATED WITH DIFFERENT A C VALUES (EQ. 6). UNTREATED A C (TOP) AND TRANSFECTED A C (BOTTOM) CONTROLS FOR ASO RESULTS ONLY. CELL VIABILITY BY MTT ASSAY.  A simple data analysis may be done to separate the treatment effects. Figure 36 (bottom graph) shows an adjusted curve for transfected samples (ASO*). When t0 untreated control cells were used for AC (Section 3.2.3, Eq. 6), the reduction in viability (ASO, top graph) was assumed to be the sum of all possible effects. When cell viabilities were recalculated using t0 transfected control cells, as the AC value, the adjusted viability values (ASO*, bottom graph) should theoretically account for the initial reduction in cell number from HSP27 knockdown. Therefore, comparison between the original control curve and the ASO* curve can act as an 61  indicator of any additional hyperthermic effect. The gaps between corresponding temperature curves in the top graph (e.g. 42C and 42C ASO) were eliminated for all temperatures in the bottom graph (e.g. 42C and 42 ASO*) of Figure 36. For transfected cells heated at 37 and 42C, this confirms that there was not an additional effect, beyond the direct effect of ASO. The only result that may suggest a “synergistic effect” was heating at 48C for 15 minutes. However, for samples heated at 48C the gap between curves of untreated (48C) and transfected (48C ASO) samples decreases over 60 minutes, which diminishes this “synergistic effect”. Also, the gap between the original control curve and adjusted ASO* curve is eliminated. Both curves exhibit a similar decreasing trend upon heating. This suggests the reduction in cell viability for transfected cells at 48C was a “combined effect” of HSP27 knockdown and heat. Although, ASO and heat kills significantly more cells than ASO alone, it does not seem to kill significantly more cells than heat alone over longer periods of time. It was not clear if heat sensitization played any role in reducing the cell viability at this higher temperature (48C).  % Cell Viability  150  37C 44C 48C 37C ASO 44C ASO 48C ASO  100  50  0 0  10  20  30  40  50  60  70  Time (mins) FIGURE 37. REPRESENTATIVE TRANSFECTION AND HEATING EXPERIMENT ON PC3 WITH 100NM ASO. CELL VIABILITY BY MTT ASSAY.  Similar experimental conditions with 100nM ASO tested on PC3, are shown in Figure 37. The results were consistent with the trends found for LNCaP. The effect on cell viability of ASO was easily seen at lower temperatures (≤ 44°C) because there is not a measurable heat effect. However, the effect of heat alone on cell viability at higher temperatures (≥ 48°C) dominates over the effect of ASO, as the cell viability between untreated and transfected 62  samples was similar. The effect of ASO and heat was not additive and it’s not clear if there is a “synergistic effect”. But there was a “combined effect”, as there is still a reduction in cell viability for transfected cells upon heat exposure. These experiments were repeated several times. Manipulation of experimental conditions, such as ASO incubation concentration, incubation time and the number of transfections, did not change the results significantly. In each case for LNCaP and PC3, there was not an obvious increase in heat sensitivity due to ASO. If there was any increased heat sensitivity it was not detected by the selected methods. Any inconsistencies with results in Figure 36 and 37 to Figure 34 may be attributed to the differences between the MTT and MTS assays and/or variability between experiments. Inconsistencies in cell viability results between experiments done with the trypan blue dye exclusion, MTT and MTS assays can be a result of the slight differences in the response of each assay to cell/proliferation activity, cell density or experimental conditions. Work by Wang et al. determined that consistency between cell viability assays is also highly dependent on the presence of interferences, as some assays may exhibit overestimation or underestimation of cell numbers [84]. 4.4 Discussion Cells typically respond to lethal treatments in two ways: arrest in the cell cycle or the initiation of protective responses. When cancer cells are exposed to a heat shock the latter type dominates, as there is a vast selection of pro-survival mechanisms [75]. Many of these mechanisms are mediated by HSP27 [69]. As a result of high heat resistance, unassisted thermal treatment of cancer must be done at high temperatures in order to be effective. Therefore, increasing the sensitivity of cancer cells to heat by gene therapy was an attractive option. Although, in photothermal therapy it is not feasible to heat tumors up to 60 minutes, extended heating was examined to get a profile of the heat response of LNCaP and PC3 cells. Both cell lines exhibit time and temperature dependent cell viability, with temperatures higher than 60C leading to complete necrosis of the cell population by 15 minutes. The knockdown of HSP27 by ASO (OGX-427) based on cell viability results was effective, as HSP27 expression can be reduced by ≥ 95% for a treatment concentration of 50nM or higher [69]. Although, cell viability results were consistent with previous reports on gene therapy targeting HSP27 [71], a direct relationship between HSP27 levels and heat sensitivity was not established. Treatment with ASO and heat can each reduce cell viability separately. However, the results did not show 63  that hyperthermia was significantly enhanced (synergistic effect) by ASO. For example, PC3 treated with 100nM ASO results in a 39% decrease in cell viability. Heating the cells for 15 minutes at 48C decreased the viability by 59 and 52% with and without treatment with 100nM ASO (Figure 37). The 7% difference between these values was not significant (p > 0.05). It is unclear if HSP 27 knockdown can enhance hyperthermia of PC3 and LNCaP. However, lowering the expression levels of HSP27 is still useful to overall treatment goals. HSP27 blockage can inhibit cell proliferation [70], enhance apoptosis of PC3 and LNCaP, delay tumor progression and increase sensitivity to other forms of treatment [71, 81]. Another added benefit is that it can inhibit the onset of thermotolerance if hyperthermic treatment is given in multiple fractions. Gene therapy targeting HSP27 may still be used in combination with photothermal treatment to increase therapeutic efficiency.  64  Chapter 5. Photothermal Treatment Studies using Gold Nanoparticles 5.1 Introduction The photothermal effect can produce heat by two distinct methods: light absorption and heat release by a photothermal agent or direct use of the optical source to generate heat [85]. Electromagnetic radiation from the ultraviolet (UV) to the infrared (IR) region may be used depending on the application. For the photothermal treatment of cancer tumors, lasers in the NIR are used to achieve deep-tissue penetration [72]. Photothermal agents are often used to increase heat production within a localized region and to lower laser intensities [74]. Gold nanoparticles activated by NIR laser irradiation can be used to deliver a controlled dose of heat to cancer cells. In this chapter, photothermal treatment was evaluated in vitro by measuring the approximate temperature increase and effect on cell viability. The best nanoparticle candidate selected from Chapter 3, the 40nm gold nanoshells, was used. A preliminary in vivo test was performed to evaluate photothermal conditions and heat generation. Laser light is monochromatic, coherent, and is easy to collimate which can provide a narrow beam of high intensity, that can propagate deep into tissue with minimal power loss and high precision [72]. Therapy with a laser requires high power densities of tens to hundreds of watts to efficiently ablate a tumor [86]. The disadvantage of strict laser therapy is its nonselectivity, as normal and cancerous cells/tissue in the path of the laser will both be damaged. Photothermal agents can improve selectivity by localizing heat generation and lowering the laser power to levels that do not damage untargeted tissue. Molecules or particles that can absorb light and release energy by nonradiative processes can be used as photothermal agents. Chromophores or dye molecules can theoretically perform this function. However, in comparison to gold nanoparticles, these choices are not ideal as they are limited by low absorbance and/or photobleaching under laser irradiation [72]. The appeal of gold nanoparticles is their efficient coupling to electromagnetic energy at SPR frequencies [13]. They absorb much stronger than conventional absorbing dyes [45, 72], and efficiently convert the absorbed light into heat [17]. Work by Richardson et al. determined the efficiency of light-to-heat conversion () for a water droplet containing 20nm gold nanoparticles to be close to one [18].They also modeled the temperature distribution in this droplet. Although, the temperature is highest around single particles there is an increase in temperature due to collective heating by the particles [18]. These properties make gold nanoparticles an appropriate candidate for medical applications. An 65  example of recent progress is the start of Stage I clinical trials using Auroshell® particles to ablate tumors in the head and neck [87]. The conditions for in vitro and in vivo model experiments are selected to mimic therapeutic conditions for treatment in humans. Model experiments must assess for the limitations that will arise in clinical practice. For photothermal treatment the absorption and scattering of light by tissue represents a treatment restriction that must be considered [88]. Major interferences in biological tissue are hemoglobin (Hb) and water (H2O), as they absorb visible and infrared light [15]. In the NIR, both species have the lowest absorption (Figure 38). Therefore, a laser wavelength in the NIR is used as it is the most biologically “invisible” and can pass through the tissue up to 10cm in depth [15] with minimal energy loss [89]. Gold nanoparticles are particularly useful photothermal agents as they can be tuned to preferentially absorb or scatter light at a specific wavelength (Chapter 2).  FIGURE 38. THE NIR WINDOW. ADAPTED FROM: [15].  In vitro studies can determine the basic requirements needed to destroy cancer cells. The photoinduced cell death of cancer using gold nanoparticles is dependent on the hyperthermic conditions generated (Figure 32). The conditions are determined by the amount of nanoparticles internalized or absorbed to the surface of cells [89, 90], laser intensity [40, 77] and total laser exposure time. Cheng et al. performed dosage studies on three cancer cell lines and found that 66  there are minimum effective dosages that are dependent on the cancer cell line [89]. Laser irradiation studies of hybrid gold nanoparticles by Kirui et al. demonstrated that 53% of colorectal cells were destroyed upon a 6 minute treatment with a power density of 5.1W/cm2 (4mm spot size) and 99% for 31.5W/cm2 [77]. The heating time is important, but the effect on cell viability is more noticeable over a larger time scale (Chapter 4). Laser treatment studies are typically performed for treatment times below 10 minutes [78, 89]. The tumor environment (in vivo) is vastly different than the cellular environment (in vitro), leading to separate conditions for photothermal treatment. With in vivo models, such as mice, laser light is directed in different ways. The laser can be transmitted from an optical fiber tip through tissue or by inserting the bare end of the fiber into the center of the tumor [72]. There are several model photothermal methodologies, as various hyperthermic conditions and experimental designs can be used. Work by Hirsch et al. demonstrated successful photothermal ablation of tumors and breast cancer cells using nanoshells, with irradiation at 820nm and laser intensities of 4W/cm2 for < 6 minutes and 35W/cm2 for 7 minutes, respectively [78]. Other considerations for treatment on tumors is the transport, accumulation and distribution of the photothermal agent in the tumor [13], and also the effect of tumor volume and penetration of the laser beam on the diffusion of heat [78]. As mentioned in earlier chapters, accumulation of gold nanoparticles may be done by a specific or nonspecific route. Nonspecific delivery of gold nanoparticles to the tumor environment can be done intravenously or by direct injection. These methods depend on accumulation in tumor tissue by means of blood circulation and/or the enhanced permeability and retention (EPR) effect [23, 26]. Tumor vasculature is physiologically distinct from normal vasculature [13] as it is characterized by a highly disorganized architecture, abnormal fluid flow and a leaky endothelium [1].These properties allow for the selective accumulation and distribution of gold nanoparticles in tumor tissue. Photothermal treatment, using gold nanoparticles and laser irradiation at 800nm, was evaluated by measuring the changes in cell viability of PC3 and LNCaP cells. The conditions of hyperthermia are directly associated to laser power, heating time and the amount of nanoparticles internalized by the cells. Therefore, these experiments can also be used as a method to confirm or refute cellular uptake results seen in Chapter 3 (e.g. gold nanorods). Determining conditions that had the greatest effect on cell viability was the target. The treatment results of the 40nm gold nanoshells were compared against the Auroshells®, as these larger particles are used in clinical practice. Finally, photothermal treatment was combined with ASO 67  gene therapy to confirm the net effect of the treatments together. A “synergistic effect” is considered as an observed increase in heat sensitization and a “combined effect” is the total effect of the individual treatments without heat sensitization (Chapter 4). A preliminary in vivo study was performed on animal models to develop basic methodology, evaluate photothermal treatment conditions and to measure a heating effect. For animal studies, direct injection was investigated. 5.2 Materials and Methods 5.2.1 Materials RPMI Medium 1640 with 25mM HEPES buffer was obtained from Invitrogen. Athymic nude mice with subcutaneous LNCaP tumors (~2cm in diameter) on the right and left hind leg [91], were obtained from Dr. Martin Gleave, Prostate Center, BC, Canada. Experimental procedures were in accordance with Canadian Council on Animal Care (CCAC). See Section 3.2.1 and 4.2.1 for additional materials. 5.2.2 Nanoparticle Heating Profiles Pure nanoparticle solutions (200µL) were irradiated using the set-up in Figure 39, without the heat source. The nanoparticle solution was exposed to a 1W laser (3W/cm2, spot size 6.5mm) for 2 and 4 minutes. The temperature was measured before and after laser exposure to determine the change in temperature (ΔT). Plots of the heating profiles are shown in Figure 41 and 42. The photothermal conversion efficiency of gold nanoparticles was calculated by Qabs/Qtot × 100% [89, 92]. Where Qabs represents the total energy absorbed by the aqueous solution containing the gold nanoparticles and Qtot represents the total energy supplied by the laser over time. The equations of Qtot and Qabs are: Qtot = (laser intensity) × (laser spot area) × (irradiation time)  (Eq. 8)  Qabs = [(MAu × CpAu) + (MW × CpW)] × (ΔT )  (Eq. 9)  Where MAu and MW are the number of moles of gold and water, CpAu and CpW are the molar heat capacity of gold and water and ΔT is the change in temperature after irradiation.  68  5.2.3 Photothermal In Vitro Heating Tests Cell Preparation PC3 and LNCaP were cultured as previously described (Section 3.2.2). This procedure requires a period of 3 Days. The experiment was performed using 40nm gold nanoshells, Auroshell® and gold nanorods. Cells were plated by adding 10,000 cells per well in a 96 well plate and incubated overnight (Day 1). Gold nanoparticles were added at the concentrations listed in Table 7 and incubated for 24 hours (Day 2). Cells prepared under similar conditions, without the addition of gold, were used to test the effect of the laser. Also, a nonirradiated control was prepared. Prior to the laser treatment (Day 3), all medium was removed and fresh RPMI medium (100 to 200µL) containing FBS (10%) was added. In later experiments, medium (100 µL) supplemented with HEPES (25mM) was added. TABLE 7. GOLD NANOPARTICLE INCUBATION CONCENTRATIONS FOR PHOTOTHERMAL EXPERIMENTS.  Nanoparticle Type 40nm Gold Nanoshell* Auroshell®** Gold Nanorod***  Incubation concentration (particles/mL) 2 x 1010 1 x 108 3 x 109  Concentrations were calculated as listed in Table 3  Description of Laser Heating Set-up The experimental set-up was developed in-house and was optimized to mimic physiological conditions. Several control experiments were performed to isolate the immediate effect of photothermal treatment by the laser. Figure 39 provides a schematic of the final set-up, showing an individual well on a 96 well plate. Preliminary experiments were done without a heat source. This was later added to adjust the background temperature, as the ambient temperature of the lab was 24C. There is an air gap between the well bottom and the heat source surface, so there is not any direct contact. Experiments with an adjusted background temperature were placed on the heat source for 10 minutes before the experiment was started. A double-pass laser scheme was assembled from the set-up in Figure 39 by raising the plate on a platform so that a reflective mirror could be inserted beneath the well.  69  FIGURE 39. ILLUSTRATION OF A SINGLE PASS EXPERIMENTAL SETUP USED FOR PHOTOTHERMAL THERAPY OF PROSTATE CANCER CELLS. ENLARGEMENT OF ONE WELL IN A 96 WELL PLATE.  A continuous wave Ti:sapphire laser with a central wavelength at 800 nm was used. The 96 well plate was positioned such that the laser spot size (D = ~6.5 mm) closely matched the diameter of the well (D = 6.38 mm). The total output power of the beam was measured with an optical power meter (Ophir Nova ΙΙ) prior to use. For example, a laser intensity of 12W/cm2 and a spot size of 6.5mm, gave a power of 4W. The duration of irradiation and the power were varied for a fixed irradiation area to determine hyperthermic conditions.  Measurement Procedure With the laser blocked, the well was aligned with the laser to ensure the well matched closely to the laser path. The initial temperature was recorded using a 33 gauge needle thermocouple (Omega Technologies) and digital readout (PeakTech). The cells were exposed to the laser light for time intervals between 2 to 15 minutes and powers of 1 to 8W. After exposure the laser was blocked and the temperature was re-measured. Each experiment was completed within 2 hours to minimize time for the cells outside of the CO2 environment and to prevent upregulation of HSPs. After laser treatment, fresh RPMI with 10% FBS (100µL) 70  was added and MTT (50µL) was added. The MTT assay and cell viability calculations were performed as previously described (Section 3.2.3). Nonirradiated cells, devoid of gold, were used as the control (AC). 5.2.4 Photothermal In Vivo Heating Tests Preparation Mice were anesthetized with methoxyflurane prior to treatment. Table 8 outlines the experiments performed. Hollow gold nanoshells were prepared by Thomas Preston, Signorell group, UBC, Canada using the procedure in Section 2.6.3. The nanoparticle solution (50µL) was injected directly into the tumor, using concentration levels 1 x 109 and 7 x 109particles/mL. TABLE 8. TEST CONDITIONS FOR PRELIMINARY ANIMAL MODEL EXPERIMENTS.  Experiment Skin Test Control Skin Test Gold Injection Tumor Test Control Tumor Test Gold Injection  Thermocouple 1 Placement Near laser spot Near laser spot Injection site, near laser spot Injection site, near laser spot  Thermocouple 2 Placement Away from laser spot Away from laser spot Base of tumor  Laser Power (W) 2.0  Base of tumor  0.5 and 1.0  2.0 0.5 and 1.0  Adjustments in laser power were done to change the overall heating conditions to minimize the visible burning of tissue.  Description of Laser Heating Set-up The experimental set-up was similar to work by Stern et al. [60]. The same laser is used as in Section 5.2.3. An 810nm laser with powers of 0.5 to 2W (spot size of 1cm) was used. Two needle thermocouples were placed in two locations to assess differences in temperature (Table 8). The placement of thermocouple 1 was buried in the tissue to avoid direct exposure with the laser. The value from thermocouple 1 represents the temperature as a result of heating by the laser alone or by the laser with the hollow gold nanoshells. Thermocouple 2 gave the temperature value of the mouse in the absence of any treatment (control).  71  FIGURE 40. THERMOCOUPLE PLACEMENT FOR SKIN (TOP) AND TUMOR (BOTTOM) TEST. BASED ON TABLE 8.  Measurement Procedure The laser was focused on the tumor or normal tissue (skin). Both experiments were done with and without the injection (control) of hollow gold nanoshells. For laser exposure with gold, the laser was applied minutes after hollow gold nanoshell injection. The temperature from both thermocouples was recorded in 1 minute intervals over a period of 5 minutes. The temperatures of the two thermocouples were used to determine the net change in temperature as a result of treatment using Eq. 10. Δ Temperature = Thermocouple 1 – Thermocouple 2  (Eq. 10)  Tissue Analysis The tumor tissue was imaged by light scattering (Section 3.2.2) and scanning electron microscopy with an energy dispersive x-ray detector (SEM-EDX, model Hitachi S-3000N) to 72  determine if nanoparticle distribution could be established. The tumor with and without gold injection were harvested by Virginia Yago, Prostate Center, BC, Canada and frozen at -80C. Tumor sections were sliced (25µm) and fixed onto microscope slides by Antonia Tsallas, Burt group, UBC, Canada. These slices could be directly imaged by light scattering. For SEM-EDX the tumor slices were dehydrated in ethanol, dried using supercritical CO2 and carbon coated by the UBC BioImaging Facility, BC, Canada. 5.3 Results 5.3.1 Nanoparticle Heating Profiles Representative temperature elevation profiles for 40nm gold nanoshells and gold nanorods in solution are provided in Figure 41 and 42. Both particle types exhibit a time and concentration dependent temperature change. These profiles match the trends in work by Liu et al. and Cheng et al. [89, 93].  FIGURE 41. TEMPERATURE PROFILES FOR TWO CONCENTRATIONS (PARTICLES/ML) OF GOLD NANORODS AT A LASER POWER OF 1W.  The calculated average photothermal efficiencies (Qabs/Qtot) for the high and low concentration of CTAB gold nanorods were 23 ± 6% and 13 ± 3%. These calculations were based on nanorod dimensions of length 65 and width 15nm. For the 40nm gold nanoshells the average efficiencies were 8 ± 1% and 5 ± 1%, respectively, for nanoshell diameter of 73  40nm with a 30nm silica core. If the conversion of light-to-heat is close to unity as literature suggests [18], the calculated photothermal efficiency may be used as a measure of the amount of total light absorbed by different particles and particle concentrations.  FIGURE 42. TEMPERATURE PROFILES FOR TWO CONCENTRATIONS (PARTICLES/ML) OF 40NM GOLD NANOSHELLS AT A LASER POWER OF 1W.  Since the photothermal efficiency increases for increasing particle concentration, it is important that a sufficient concentration of nanoparticles be present inside cancer cells (Chapter 3) and particles that have high photothermal efficiency be used to have the best therapeutic effect. Some more detailed heat profile studies may be found in literature. A comparative study by Cheng et al. showed that for equivalent particle dosages (8.36 x 108 particles/100µL) of ~125.4nm bare gold nanoshells and coated nanorods with an aspect ratio of ~3.9, nanoshells had a slightly higher photothermal efficiency [89]. However, the difference in efficiency was < 6%. Gold nanorods and 40nm gold nanoshells in Figures 41 and 42 most likely had comparable efficiencies, as for concentrations of 8.8 x 10 10 and 7.0 x 1010particles/mL the calculated efficiencies were 13% and 8% respectively.  74  5.3.2 Photothermal In Vitro Heating Tests Development of Experimental Procedure Some photothermal studies in literature perform the cell heating with key procedural differences. Two main examples are: irradiation of the cells without medium (e.g. cell on a cover slip) [94] and using a laser spot size that is considerably smaller than the area of plated cells [77, 89, 95]. The set-up in Figure 39 includes a small volume of media (100 to 200µL) to prevent cellular stress unrelated to thermal treatment. However, keeping cells in media will results in losses in heat. Some experiments that use a laser spot smaller than the surface area of cells, often use cells that are overgrown or at 100% confluence, and measure the difference in viability at the treatment spot only. In Figure 39 a spot size that matched the well diameter was chosen to ensure the majority of the cell population was exposed to the laser. Therefore, cells could be grown to a healthier confluence of 70 to 90% before treatment and all cells were evaluated in the cell viability determination. Any inconsistencies in plating should not affect cell viability results as the laser spot size matches the well size. Several preliminary experiments were performed at a laser power of 1W at an ambient temperature of 24°C to configure the set-up and to determine the initial affect of the laser on the cell lines. PC3 and LNCaP cells irradiated up to 10 minutes with a 1W laser exhibited an increase in temperature of 2C with no effect on cell viability. The increase in temperature was attributed to light absorption of the cell media. Cells incubated with 40nm gold nanoshells resulted in a minor temperature increase with a 1W laser. This may be the result of a low nanoparticle internalization, insufficient laser power or high heat loss. As the incubation concentrations demonstrated good uptake in Chapter 3, the latter conditions were changed. The laser power was increased to 4W and the volume of medium was reduced from 200 to 100µL. Several control experiments were performed to determine the effect of the laser on cells, devoid of gold nanoparticles, for laser irradiation for 2, 4, 6, 8 and 10 minutes. Both cell lines were unaffected by the increase in laser power to 4W, as their viability remained close to 100%. Temperature increases did not exceed 5C for up to a 10 minute laser exposure. These conditions improved the temperature change for samples containing 40nm gold nanoshells to 12C. Although a net change in temperature of ~7C (12C minus 5C) was observed due to the collective heating of 40nm gold nanoshells, cell viability was not significantly different (p > 0.05) from nonirradiated control samples. This was reasoned because final treatment 75  temperatures were at 36°C (24C plus 12C), as there is a drop in cell temperature from physiological to ambient temperatures (24C) in the laboratory. If the cell temperature were maintained at 37C, the temperature effect of the laser and gold nanoparticles could be 49C. An experiment performed with a background temperature of 37C did generate a final temperature of ~49C, which resulted in a 30% decrease (p < 0.05) in cell viability (Figure 43). This value is consistent with the trends seen in Figure 32.  PC3  % Cell Viability  125 100 75 50 25 0 0 m in  4 m in  6 m in  Laser Exposure FIGURE 43. REPRESENTATIVE PHOTOTHERMAL EXPERIMENT ON PC3 INCUBATED WITH 40NM GOLD NANOSHELLS. IRRADIATED WITH A 4W LASER WITH A 37C BACKGROUND TEMPERATURE.  In order to further study the effect of treatment on cell viability, higher temperatures were needed. However, a laser power of 4W was the maximum that could be generated for the current single-pass laser scheme. Increasing the laser power was attempted by configuring the set-up to accommodate for a double-pass of the 4W laser beam to generate 8W. This created several difficulties. The background temperature could not be well maintained because placing the 96 well plate on an elevated platform, to insert a reflective mirror below, resulted in a increased gap between the heat source and plate. Also, the power of the laser could not be measured. There are losses in laser power due to absorption and scattering of the medium and the plate, so the laser power was expected to be < 8W. Rather than adjusting laser power, modifying the background temperature seemed more appropriate to increase the final temperature a few degrees. The finalized set-up consisted of a 4W laser irradiation with an elevated background temperature of 42C creating final temperatures up to 54C with 76  40nm gold nanoshells. PC3 and LNCaP did not exhibit any adverse effects from these background settings.  FIGURE 44. REPRESENTATIVE CONTROL EXPERIMENT ON LNCAP (LEFT) AND PC3 (RIGHT) WITHOUT GOLD NANOPARTICLES. IRRADIATED WITH A 4W LASER WITH A 42C BACKGROUND TEMPERATURE.  Background temperatures of 24 to 42C did not significantly affect cell viability (p > 0.05) for both cell lines for all time points. Figure 44 shows the 6 and 10 minute laser exposure for both cell lines at a background temperature of 42°C. Also, both cell lines remained healthy in the absence of a CO2 environment for the duration of the experiment (2 hours). However, for experiments using the finalized experimental set-up, medium containing HEPES buffer was added to help maintain optimal pH for cells while outside a CO2 environment. In general, a maximum temperature is reached between 2 to 4 minutes as equilibrium is established with the surroundings. Temperatures before and after laser exposure were measured to determine the change in temperature (ΔT). However, it is expected that the second measurement is an underestimate of the true value, because as soon as the laser is blocked the temperature of the 100µL of solution decreases. Therefore, it is possible that the actual final temperature is a few degrees greater than what was recorded. Photothermal Treatment with Gold Nanorods Treating PC3 and LNCaP cells with a concentration of 3 x 109particles/mL (Table 7) of gold nanorods and the 4W laser resulted in no significant change in cell viability (Figure 45) and a minimal change in temperature. For laser treatment times of 6 and 10 minutes on LNCaP, control samples exhibited a 3.9 ± 0.4 and 3.4 ± 0.3C change in temperature. LNCaP 77  treated with gold nanorods exhibited a 4.6 ± 1.0 and 5.3 ± 0.9C change respectively. Laser treatment on PC3 exhibited similar temperature results. These temperature changes were most likely due to the heating from the laser alone. All cell viability results, except the 6 minute laser exposure on LNCaP (13% difference), were not significantly different (p > 0.05).  FIGURE 45. REPRESENTATIVE PHOTOTHERMAL EXPERIMENT ON LNCAP (LEFT) AND PC3 (RIGHT) INCUBATED WITH GOLD NANORODS. IRRADIATED WITH A 4W LASER WITH A 42C BACKGROUND TEMPERATURE.  Photothermal treatment with nanorods established that the absence of scattering in images in Section 3.3.3 was in fact due to lack of cellular uptake of the PEG coated nanorods. However, successful treatment has been documented using nanorods as they do have favorable properties, such as good photothermal efficiency. Work by El - Sayed et al. used anti-EGFR conjugated gold nanorods to treat oral cancer cells and found intensities of 19W/cm2 of a 800 nm laser light for 4 minutes induced irreversible cell injury [94]. This treatment does not rely on the internalization of nanorods, but the specific binding of nanorods to the surface of the cancer cells.  Photothermal Treatment with Gold Nanoshells Photothermal treatment with 40nm gold nanoshells (NS40) and Auroshells® (NSAS), using concentrations outlined in Table 7, were compared. The photothermal efficiency (Section 5.2.2) of pure solutions of 40nm gold nanoshells and Auroshells® were calculated based on the ICP-MS concentration results (2.6 x 10-4 and 9.2 x 10-5 molAu/mL). At an irradiation power of 4W for 1 minute and ΔT values of 59 and 29°C, the efficiencies were 21% and 10%, respectively. Although the particle concentrations are not exactly equivalent, 78  the measured absorbance of the nanoparticle solutions was close in value. Using this information, it can be assumed that the amount of absorbing gold atoms per nanoparticle solution is approximately equal. Therefore, for equivalent light absorption the thermal conversion is almost double for the smaller nanoshells. The higher efficiency for 40nm gold nanoshells is consistent with the fact that smaller particles generate and dissipate more heat [26].  FIGURE 46. REPRESENTATIVE PHOTOTHERMAL EXPERIMENT ON LNCAP (LEFT) AND PC3 (RIGHT) INCUBATED WITH 40NM GOLD NANOSHELLS (NS40) AND AUROSHELL® PARTICLES (NSAS). IRRADIATED WITH A 4W LASER WITH A 42C BACKGROUND TEMPERATURE.  The cell viability results for treatment with NS40 and NSAS are shown in Figure 46 for a 4W laser exposure. A summary of the results is given in Table 9. Irradiation at this laser power with an elevated background temperature of 42C resulted in an increase of 12C for samples incubated with 40nm gold nanoshells and 9C with Auroshells®, for both PC3 and LNCaP. Therefore, net temperature increases due to the particles were 7 and 4C, respectively. The change in cell viability in Table 9 is the decrease in value with respect to the nonirradiated control (100%). The greatest cell viability decrease for LNCaP and PC3 was 54 and 71%. This proves that an adequate amount of gold nanoparticles were internalized by the cells to generate a heating effect.  79  TABLE 9. SUMMARY OF CELL VIABILITY DECREASES AND CHANGES IN TEMPERATURE FOR PHOTOTHERMAL EXPERIMENTS WITH 40NM GOLD NANOSHELLS AND AUROSHELL® PARTICLES IN FIGURE 46.  Laser Irradiation (minutes) 6 10  Sample NS40 NSAS NS40 NSAS  Δ Temp (C) 12 ± 1 8±1 11 ± 3 9±1  LNCaP Δ Cell Viability (%) 54 27 44 35  p value < 0.05 < 0.05 < 0.05 < 0.05  Δ Temp (C) 12 ± 1 9±1 12 ± 1 9±2  PC3 Δ Cell Viability (%) 68 39 71 60  p value < 0.05 > 0.05 < 0.05 < 0.05  Photothermal therapy with the 40nm gold nanoshells resulted in a larger effect on cell viability and exhibited a greater change in temperature suggesting a higher therapeutic efficiency as compared to Auroshells®. This may be a result of the higher photothermal efficiency, greater cellular uptake or both. Treatment performed on PC3 did demonstrate a greater effect on cell viability as compared to LNCaP. This may be a result of LNCaP having more effective survival mechanisms or potentially a greater gold nanoparticle uptake for PC3 (Chapter 3). However, the temperature data in Table 9 does not correlate well with increased cellular uptake, as there was not an observed difference in temperature change (ΔT) between the cell lines.  Photothermal Treatment with 40nm Gold Nanoshells Combined with ASO Gene Therapy Gene therapy with HSP27 ASO was found to reduce cell viability (Chapter 4) by inhibiting cell proliferation and promoting apoptosis [70]. Therefore, the combination of photothermal treatment with ASO gene therapy may still create a “combined effect” to destroy cancer cells. The results of the treatments are shown in Figure 47.  80  FIGURE 47. REPRESENTATIVE PHOTOTHERMAL EXPERIMENT ON LNCAP (LEFT) AND PC3 (RIGHT) TREATED WITH 100NM ASO AND 40NM GOLD NANOSHELLS. IRRADIATED WITH A 4W LASER WITH A 42C BACKGROUND TEMPERATURE.  Treatment with 100nM HSP27 ASO resulted in an initial decrease in cell viability of ~50% and ~39% for LNCaP and PC3. Treatment of ASO transfected samples, in the absence of gold nanoparticles, with the laser for 6 and 10 minutes had no additional effect on cell viability or temperature. Treatment with ASO and 40nm gold nanoshells followed by laser irradiation resulted in a net ~76% decrease in viability for PC3 and up to 67% decrease for LNCaP (Table 10). TABLE 10. SUMMARY OF CELL VIABILITY DECREASES AND CHANGES IN TEMPERATURE FOR PHOTOTHERMAL EXPERIMENTS WITH 40NM GOLD NANOSHELLS AND 100NM ASO IN FIGURE 47.  Sample ASO ASO + NS40 ASO + NS40  6  Δ Temp (C) 11 ± 1  LNCaP Δ Cell Viability (%) 50 64  10  11 ± 1  67  Laser Irradiation (minutes)  < 0.05 < 0.05  Δ Temp (C) 9±1  PC3 Δ Cell Viability (%) 39 76  < 0.05 < 0.05  < 0.05  10 ± 1  76  < 0.05  p value  p value  When the NS40 results of Table 10 to Table 9 are compared, there is not a significant difference between NS40 and ASO + NS40 treatments for all samples, which is consistent with trends seen in Chapter 4. The effect of ASO and heat was larger than the effect of ASO alone, but not the effect of heat alone. It was not clear if ASO affected the heat sensitivity of PC3 and LNCaP. However, the combined treatment at temperatures of ~53C, resulted in the destruction of up to 76% of the cell population. Table 10 compared to heating results in 81  Chapter 4 may suggest a “synergetic effect”, but any inconsistencies in cell viability may be due to potentially higher temperatures generated from heating with the laser and gold nanoparticles. Although estimated temperatures of 49 to 54C were reached in the 100µL of medium, this may not be the true value as discussed previously. Also, temperatures at or near the cell may be higher than the bulk medium, as a result of gold nanoparticle internalization in the cells. 5.3.3 Photothermal In Vivo Heating Tests: Preliminary Mouse Model Study The efficacy of gold nanoparticles is well recognized in cancer treatment models, especially in mice [60, 96]. Many of those studies commonly use intravenous administration of nanoparticles. In this work, the main focus was to develop methodology for direct injection, test the experimental conditions on healthy and tumor tissue, establish if a measurable temperature change was possible and determine the approximate distribution of the gold nanoparticles in tumor tissue. The NIR window is known to be the range of light that is biologically “invisible” permitting optimal penetration [15][74]. The effect of laser light on normal tissue was first observed to verify this. Figure 48 demonstrates the effect of the laser with and without hollow gold nanoshells.  FIGURE 48. REPRESENTATIVE PHOTOTHERMAL EXPERIMENT ON MOUSE MODEL. TESTED ON HEALTHY TISSUE. IRRADIATED WITH A 2W LASER WITH AND WITHOUT DIRECT INJECTION OF HOLLOW GOLD NANOSHELLS.  82  The control in Figure 48 represents the effect of the laser alone on the skin of the mouse. There was some absorption by the skin at a laser power of 2W. The average change in temperature was 11 ± 1C, as determined by Eq. 10, for a base temperature (Thermocouple 2) of 36°C. This value was unexpectedly high, suggesting the laser power may be too high or there may be some direct heating of the thermocouple by the laser. Injection of hollow gold nanoshells, with a concentration of 7 x 109 particles/mL, into the skin gave an average temperature change of 25 ± 3C (base temperature: 33°C) and a noticeable burning of the skin after 3 minutes laser exposure. The presence of the gold nanoparticles raised the temperatures well above the damage threshold necessary to induce tissue damage [78]. There was a substantial difference in temperatures generated (~14C), with and without gold nanoparticles. Therefore, at an optimal laser power, maximum laser penetration with minimal damaging effect with the laser alone is possible. The laser power was reduced to 0.5W for experiments on tumors resulting in a very minimal effect on control and gold injected samples. A representative treatment with a laser power of 1W and reduced hollow gold nanoshell concentration treatment is shown in Figure 49.  FIGURE 49. REPRESENTATIVE PHOTOTHERMAL EXPERIMENT ON MOUSE MODEL. TESTED ON TUMOR TISSUE. IRRADIATED WITH A 1W LASER WITH AND WITHOUT DIRECT INJECTION OF HOLLOW GOLD NANOSHELLS.  83  A 1W laser treatment of tumor tissue resulted in a higher temperature change in respect to the 2W treatment on normal tissue. The average change in temperature was 17 ± 1C (base temperature: 31°C), calculated by Eq. 10, in the absence of hollow gold nanoshells. The increased temperature may be the result of higher absorption due to the higher blood content of the tumor. Tumors were slightly overgrown and were visibly darker in color (Figure 40), and therefore may absorb more NIR energy. Also direct heating of the thermocouple by the laser may also be possible. For a localized injection of hollow gold nanoshells with a concentration of 1 x 109particles/mL, an average temperature change of 22 ± 1C (base temperature: 33°C) was measured. Burning of the tissue was also observed after 4 minutes of laser exposure. In both cases, high enough temperatures were generated to induce damage to the tumor [78]. There are several issues that need to be addressed before reliable and systematic in vivo measurements can be made. Firstly, thermocouple placement must be improved to standardize location and depth to minimize contact with the laser, as it was suspected that there may be some interference. Also, the injection location and the orientation of the tumor to the laser between different tumors treated were inconsistent. It was unclear in the set-up by Stern et al. how these issues were addressed [60]. Also, there were no controls set into place about the effect of tumor volume on particle distribution and heating. Despite this, there are documented successes in treating tumors by photothermal therapy. For example, O’Neal et al. treated mice with colon cancer tumors using a gold nanoshell solution by tail vein injection [96]. Treatment was performed with a 808nm laser at 4W/cm2 for 3 minutes which increased surface temperature at the tumor site, to ~50°C. Monitoring for 90 days revealed all mice were healthy and tumor free. Further exploring direct injection of gold nanoparticles into tumors is also attractive, as it can shorten treatment times because particles do not need time to accumulate. The treatment conditions from the in vitro to the in vivo case were different especially in respect to laser power. Powers generally lower than 1W can be used to generate temperatures of over 50C in vivo. This occurs because there are differences in thermal diffusion [93]. The distribution of the hollow gold nanoshells in tumor tissue was investigated by light scattering and SEM-EDX. Several slices of tumor tissue were examined. Slices from a plain tumor were chosen at random throughout the tumor volume as a control for comparison against tumors with gold. The plain tumor did exhibit some scattering (Appendix A.2.1) 84  which may be attributed to the microstructure of the tissue [88]. Slices from the tumor injected with gold were selected surrounding the injection site, as this most likely had the highest concentration of hollow gold nanoshells. The slices exhibited scattering as well (Appendix A.2.1), similar to that of the plain tissue. It was not possible to determine if there was additional scattering from the hollow gold nanoshells. When the relative intensity of plain tumor slices and tumor slices with gold were compared, the scattering was not visibly different suggesting the scattering of tissue may be too high to image properly. Therefore, the distribution of hollow gold nanoshells could not be determined with certainty by this method. The SEM-EDX images did not show the presence of hollow gold nanoshells (Appendix A.2.2). However, this may be a result of the preparation method, as dehydration in ethanol may have washed away any hollow gold nanoshells on the surface. The temperature results do show a measurable difference between tissue with and without gold, but it is unknown how dispersed the particles were. A study by Hirsch et al. performed photothermal therapy on tumors by direct injection of nanoshells [78]. They determined that the nanoshells had a diffuse distribution throughout the tumor within minutes of injection. This was confirmed by examination of the heating profiles by MRI and the histological analysis of tissue samples. The injection protocol in this present work matches closely to that of Hirsch et al., so most likely the tumor can be considered to have a good nanoparticle distribution that was not measured by light scattering or SEM-EDX. 5.4 Discussion The photothermal treatment of LNCaP and PC3 in vitro using gold nanoshells was successful as controlled temperatures of 49 to 54C can be produced. Similar temperature ranges can be generated in vivo using much lower laser powers as a result of the lower thermal diffusion of tissue [93]. Depending on the classification on time length of heating, this temperature range can be classified as a moderate to high hyperthermic condition (Table 6). A decrease in cell viability of up to 70% was obtained when 40nm gold nanoshells were combined with a relatively low laser power of 4W at a background temperature of 42C. The net increase in temperature due exclusively to gold nanoshells internalized by cells, in 100µL of medium, was 7C. Changing treatment time from 6 to 10 minutes did not affect cell viability significantly. When photothermal therapy was combined with ASO gene therapy the cell viability matched closely to result trends seen in Chapter 4. Each therapy has an independent 85  effect on cell viability and demonstrates a “combined effect” when the treatments were performed together. However, the overall effect was not significantly greater (p > 0.05) than the effect of heat alone. For example, a 100nM ASO treatment on PC3 results in a 39% decrease in cell viability and photothermal treatment for 6 minutes results in a 68% decrease. When the treatments were combined a 76% decrease was achieved. There might be a slight “synergistic effect” between the two treatments at higher temperatures, but the selected methods in this study do not provide information on the individual effects. The MTT assay cannot distinguish between cells killed by apoptosis from ASO, necrosis from heat or cell death from increased heat sensitization as a result of ASO. It can only analyze the net effect on cell viability. The use of gold nanoparticles as photothermal agents is favorable as they have a high efficient coupling to light at SPR frequencies [13] resulting in good light absorption and lightto-heat conversion, and subsequently dissipation of heat. The photothermal efficiency of particles used is dependent on concentration and particle type. Work by Cheng et al. revealed that nanoshells have a slightly higher efficiency than nanorods [89]. Comparative work shown in this chapter suggests the smaller 40nm gold nanoshells have a higher efficiency than the larger Auroshells®. This result is supported by the fact that the 40nm nanoshells had the greatest impact on cell viability when used in photothermal therapy, as higher temperatures could be reached. The better efficiency of the smaller nanoshells may be a result of greater photothermal efficiency, higher cellular uptake or both. Nanorods did not exhibit a photothermal effect due to low nonspecific uptake, confirming light scattering results in Chapter 3. Finally, results seemed to be more pronounced for PC3 than LNCaP under treatment conditions in this chapter.  86  Chapter 6. Summary and Future Work 6.1 Summary Multimodal or combination therapy represents a new approach in fighting disease, such as cancer. As most existing treatment options may not be completely effective individually and/or lead to recurrences, combining strategies may be the best alternative to improve treatment outcomes. Selection of the modes of therapy should be based on developing the least invasive and most efficient total treatment. The original research proposal (Section 1.3) outlined the development of a formulation of gold nanoparticles, taxane drugs and ASO combining the action of thermal, chemo and gene therapy for localized prostate cancer treatment. The key objectives of this study were the investigation of photothermal therapy by gold nanoparticles and laser irradiation, and HSP27 ASO gene therapy. A selection of gold nanoparticles with desired size, stability and optical properties were investigated to act as an agent for photothermal therapy. The main particle criterion was absorption in the NIR and a size under 100nm. Gold nanoshells, gold nanorods, gold corecorona nanoparticles and hollow gold nanoshells were evaluated by their simplicity in synthesis, overall properties (Chapter 2) and cellular uptake into the prostate cancer cell lines, PC3 and LNCaP (Chapter 3). Small gold nanoshells (40nm) exhibited the most straightforward synthesis and had the best cellular uptake observed in light scattering images. As larger nanoshells are currently being studied in clinical trials, 150nm nanoshells (Auroshell®) were purchased from Nanospectra Inc. and used as a standard to compare cell internalization and photothermal treatment results in vitro. Overall, hyperthermic conditions were generated and easily controlled using gold nanoshells and a laser to give a therapeutic dose of heat (Chapter 5). The 40nm gold nanoshells and Auroshells® proved to be both optically and thermally robust as they were able to endure treatment times and temperatures. Smaller gold nanoshells exhibited better internal distribution throughout the cell and had a higher photothermal efficiency of 21%, compared to Auroshells® at 10%. This supports their greater impact on cell viability and higher change in temperature upon laser irradiation. The net temperature change due to the collective heating of 40nm gold nanoshells internalized by cells was 7C. For Auroshells® a 4C temperature change was achieved. Photothermal therapy with 40nm gold nanoshells resulted in up to a ~70% decrease in cell viability generated by a laser power of 4W and elevated background temperature of 41C. It is uncertain if the addition of ASO gene therapy can sensitize the cancer 87  cells to hyperthermia (Chapter 4). However, treatment with ASO alone does exhibit a concentration dependent effect on cell viability which may be useful to overall treatment goals as a supplement to the treatment of cancer cells/tumors. ASO will be especially important when chemotherapy is included in the total treatment, as HSP27 knockdown is known to sensitize cancer cells to drug treatment [69, 80, 81]. 6.2 Future Work The studies presented in this thesis have shown detailed synthesis of a selection of gold nanoparticles and the systematic selection of a nanoparticle candidate based on its properties and efficiency to be used as a photothermal agent. However, in respect to the synthesis of the particles there is some uncertainty to the actual concentration of particles. There are several documented ways to calculate concentration. Some examples shown in this work were calculation using ICP-MS results [19, 43] and the amount of reactants [34]. Another example, is calculating the concentration based on Mie Theory [22], as was supplied for the Auroshell® particles. However, there have not been any studies done in literature to compare these methods and evaluate the accuracy of these values generated, as they each give a different concentration value. Each of these methods is based on several assumptions and estimations to determine a concentration value. If a standardized method of concentration determination were used it may improve results comparison, between different particle types and particle concentrations, and allow quantification of the photothermal effect for given treatment conditions. Values for cellular uptake (particles/cell) may also be associated with treatment concentrations (particles/mL). Further studies may be done to also study the thermal stability of particles, as many may suffer from structural collapse when used as a photothermal agent. Examining the effect of changing particle properties on treatment results would be useful to further evaluate the overall feasibility of the particles. Targeting of HSP27 did not seem to result in sensitization of LNCaP and PC3 to heat. However, this result may be further examined if cell viability is analyzed by techniques that can provide more information and/or separate apoptotic and necrotic cell death. HSP27 knockdown is known to decrease cell death by apoptosis and photothermal treatment will cause death by necrosis. Understanding the relative contributions of each may help determine if there is a “synergistic effect”. An example of a technique that may be used measure the proportion of cell death due to necrosis and apoptosis is double staining with Annexin V fluorescein 88  isothiocyanate (FITC) and propidium iodide (PI) [77]. Apoptotic cells are PI negative and Annexin V positive, while necrotic cells are positive for both PI and Annexin V. Another option for the gene therapy approach would be to knockdown the expression of an HSP that will dramatically sensitize cancer cells to heat, such as HSP70 [83] or HSP72 [80]. This would help the therapeutic efficiency and possibly lower the treatment temperature. However, the addition of another ASO, to those already targeting HSP27 and clusterin, may overcomplicate the therapeutic strategy. The photothermal studies may be improved by using a more accurate and responsive method to measure changes in temperature for in vitro and in vivo studies. In the present work, thermocouples were used to measure temperature. However, to get a better temperature profile and thermal dosimetry, especially throughout tumor volumes, thermal data recorded from an IR thermographic camera [97] or MRI [78] may be more appropriate. This will also eliminate issues with inconsistent thermocouple placement and interference of the laser with the thermocouple. There are several other aspects that need to be investigated before a complete treatment formulation can be developed as outlined in the original research proposal. The added effect of the chemotherapy drugs, paclitaxel and/or docetaxel, must be investigated to see if there is a “synergistic effect” with photothermal and gene therapy. Once all three components have been assessed, relative concentrations of each must be determined and the delivery mechanism of the taxane drugs and ASO must be established, to produce therapy conditions that will result in successful prostate cancer treatment. Standardized methodology for tests in animal models must be developed to evaluate select formulations. Protocols must also be put in place to analyze tissue after treatment and also to monitor tumor volumes over a period of time to determine efficacy.  89  References [1] [2] [3]  [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]  Pietronave, S.; Iafisco, M.; Locarno, D.; Rimondini, L.; and Prat, M. Journal of Applied Biomaterials & Biomechanics, 2009, 7, 77-89. DeVita, V. T.; Hellman, S.; and Rosenberg, S. 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TRANSMISSION (LEFT) AND SCATTERING (RIGHT) IMAGES OF PLAIN TUMOR TISSUE.  FIGURE A7. TRANSMISSION (LEFT) AND SCATTERING (RIGHT) IMAGES OF TUMOR TISSUE WITH HOLLOW GOLD NANOSHELLS INJECTED.  97  A.2.2 SEM-EDX Image of Tumor Tissue  FIGURE A8. SEM-EDX OF TUMOR TISSUE WITH HOLLOW GOLD NANOSHELLS INJECTED.  98  

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