以金奈米棒之光熱效應破壞癌細胞之活體研究

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(1)國立臺灣師範大學 . . 物理系 . 碩士論文以金奈米棒之光熱效應破壞癌細胞之活體研究 . In vitro and in vivo photothermal destruction of tumors by gold nanorods and laser irradiation. 劉耀文 ( Bruce Yao-Wen Liu ). 指導教授: 賈至達博士陳洋元博士 . 中華民國一○一年六月 .

(2) Abstract Gold nanorods (AuNRs) have tunable plasmon-resonant absorption in near-infrared (NIR) region and excellent photothermal transduction, making them attractive for the next generation of cancer therapy.. In this study, AuNRs with a longitudinal. absorption peak at 800 nm were prepared by one-pot synthesis method, and are further characterized by absorption spectra, electron microscopy, zeta potential and inductively coupled plasma mass spectroscopy.. Both in vitro and in vivo approaches were. performed to evaluate AuNRs’ therapeutic efficacy.. According to. dual color fluorescent staining imaging, cell destruction greatly depends on influence of energy and the amount of nanorods taken up. Laser-induced heating was applied to investigate the therapeutic effect of AuNRs, where the alteration in tumor size and vascular development was examined in the mouse ear. In the experiments, murine breast tumor cells were first introduced into the ears of Balb/c female mice.. As the tumors grew to about 30. mm3 in size at the 10th day, AuNRs (~10 µg) were intratumorally injected into the ears directly, followed by employment of laser.

(3) irradiation.. The. results. of histological section. and. size. measurements indicated that the photothermal effects of AuNRs are significantly destructive to the tumor development and may lead to final removal of the solid tumor.. These results suggested that. AuNRs have great potential in future clinical phototherapeutic applications.. Key words: Surface plasmon resonance, gold nano rods, photothermal therapy.

(4) 中文摘要因為金奈米棒可吸收近紅外光以及表面電漿共振之特性，讓金奈米棒具有很棒的光熱轉換效率，此高效率產生之熱量可以有效的殺死癌細胞。藉由一步合成法，可以有效率的合成出吸收峰值落於 800 nm 的金奈米棒，藉由紫外光-可見光光譜儀、穿透式電子顯微鏡、Zeta Potential 以及 ICP-MS 測量儀之檢測，可以清楚了解金奈米棒的特性以及江奈米棒定量。在細胞以及活體實驗上均可觀察到利用金奈米棒所進行之光熱作用在治療上的效果；在細胞實驗上，藉由染劑的觀察，可以有效判斷出癌細胞的存活狀態，實驗結果大致可得到以下結論，金奈米棒被細胞吞噬之數量以及雷射之能量為影響細胞存活的最大變因；在活體實驗中，由於選用老鼠耳朵當做實驗對象，因此癌細胞增長所導致的血管新生現象也可以很容易就被觀察到，活體實驗大致流程為下：將癌細胞植入老鼠左右耳上，約到第十天左右，腫瘤體積約為 30 mm3 ，此時將 ~10 µg 金奈米棒注射於腫瘤處，最後進行雷射之光熱治療。從組織切片之結果以及腫瘤體積量測之生長曲線結果判斷，可以觀測到金奈米棒受雷射激發後所產生之光熱效應，可以嚴重的破壞癌細胞的增長甚至是將腫瘤的大部分患部移除。這些結果可以看出光熱治療此機制在未來臨床應用上具有很大的潛力。關鍵字：表面電漿共振，金奈米棒，光熱治療 .

(5) TABLE OF CONTENTS PAGE Abstract 中文摘要 Acknowledgements Table of contents ............................................................................. i List of figures.................................................................................. iii Chapter 1. Introduction ................................................................ 1 . 1.1 Properties of AuNRs ............................................................... 2 1.1.1 Surface plasmon resonance ........................................... 3 1.1.2 Quantum confinement effect........................................... 4 1.2 Introduction of cancer.............................................................. 6 1.2.1 Differences between cancer and normal cells ................ 7 1.2.2 Metastasis of cancer cells............................................... 9 1.3 Cancer therapies ................................................................... 11 Chapter 2. Experimental details ................................................ 18 . 2.1 Chemicals ............................................................................. 18 2.2 Instruments ........................................................................... 20 2.3 Preparation of AuNRs ........................................................... 21 2.3.1 Synthesis of AuNRs ...................................................... 21 2.3.2 Surface modification with polystyrenesulfonate ............ 24 2.3.3 Characterizations of AuNRs ......................................... 26 . i .

(6) 2.3.3.1 UV-visible spectroscopy........................................... 26 2.3.3.2 Transmission electron microscope .......................... 28 2.3.3.3 Zeta potential measurement .................................... 29 2.3.3.4 Inductively coupled plasma mass spectroscopy ...... 32 2.4 In vitro experiments ............................................................... 33 2.4.1 Cell line ......................................................................... 33 2.4.2 Cell culture .................................................................... 34 2.4.3 Cell viability assay (MTT Assay) ................................... 35 2.4.4 In vitro photothermal therapy ........................................ 37 2.4.4.1 Introduction to fluorescent Dyes .............................. 38 2.4.4.2 Confocal microscope and two-photon luminescence imaging .................................................................... 40 2.5 In vivo experiments ............................................................... 44 2.5.1 Animal model ................................................................ 44 2.5.2 Anesthesia .................................................................... 46 2.5.3 In vivo photothermal therapy ........................................ 46 2.5.3.1 Experimental processes........................................... 46 2.5.3.2 Microscope system .................................................. 48 2.5.3.3 Estimation of tumor size ......................................... 48 2.5.3.4 Histological sections ................................................ 49 Chapter 3. Results and discussions ......................................... 50. 3.1 Characterizations of gold nanorods ..................................... 50 3.2 In vitro cancer cell photothermolysis mediated by AuNRs .. 56 3.3 In vivo tumor photothermal therapy....................................... 63 3.3.1 Power density test with the ears model ........................ 63 3.3.2 Tumor growth curve ...................................................... 64 3.3.3 Histological study .......................................................... 69 Chapter 4. Conclusions .............................................................. 71 References ................................................................................... 73. . ii .

(7) LIST OF FIGURES Page. Figure. 1.1 The schematic illustration of plasmon oscillation in a gold nanoparticle under light irradiation. The resonance of electrons across the particle can be induced by the electromagnetic field of the light. (Unpublished material of Cheng-Lung Chen) ............ 4 2.1 Procedure of one-pot synthesis of gold nanorods .................. 25 2.2 Absorption of bio-organism ..................................................... 26 2.3 Visible spectrum .................................................................... 27 2.4 Comparisons of microscope ................................................... 28 2.5 The structure of TEM .............................................................. 29 2.6 Schematic of zeta potential measurement ............................ 31 2.7 Zeta potential cell .................................................................... 31 2.8 Schematic of ICP-MS measurement ..................................... 33 2.9 Preparation process of PSS-AuNRs with two different concentrations ......................................................................... 33 2.10 Molecular of structure (MTT) ............................................... 35 2.11 Absorption and fluorescence emission spectra of YO-PRO-1 bound to DNA ...................................................................... 38. 2.12 Absorption and fluorescence emission spectra of propidium iodide bound to DNA .......................................................... 40 2.13 Fluorescent images of destroyed cell(a) Transparent pattern. . iii .

(8) (b) After the excitation, the emission signal (green) by YO-PRO-1 was detected. (c) After the excitation, the emission signal (red) by. PI was detected. (d) Emerge. signals of (a), (b), (c) ............................................................ 40 2.14 The difference between conventional and confocal microscope. The image indicates that the confocal image has better resolutions than the conventional image. ................. 41 2.15 Beam path of the excitation and emission light in a confocal laser-scanning microscope ................................................ 42 2.16 Differences between single photon and two-photon excitation ............................................................................................... 43 2.17 Two-Photon Iuminescence Images of gold nanordods. (a) Only the emission signal of AuNRs. (b) Signal by merge (a) and transparent light. ........................................................... 44 2.18 Schematic of the ear structure ............................................ 45 2.19 Image of the histologic section of the mice ear ..................... 45 2.20 Set-up of the in vivo photothermal therapy ......................... 47 2.21 Schematic of the in vivo photothermal therapy ................... 47 3.1 (a) TEM image of PSS-AuNRs. (b) High resolution TEM image a single AuNR ......................................................... 51 3.2 Absorption spectroscopy of CTAB- and PSS-coated AuNRs . 51 3.3 (a) The zeta potential of CTAB-AuNRs ................................. 52 3.3 (b) The zeta potential of PSS-AuNRs ................................... 53 3.4 (a) Calibration data of Au ........................................................ 54 3.4 (b) Measured data of PSS-AuNRs .......................................... 55. . iv .

(9) 3.4 (c) The results of ICP-MS measurement. The blue points are the standard curve of calibration, and the red points are the results of the analysis. ........................................................... 55 3.5 Cell viability of EMT-6 cells exposed to CTAB- and PSS-coated AuNRs after (a) 12 h, (b) 24 h and (c) 48 h incubation,respectively. The viability of the cells was normalized with respect to a media-only control.................... 57 3.6 Photothermolysis of the EMT-6 tumor cell triggered by PSS-AuNRs (N~30–40 clusters) under energy fluence 24.2 mJ/cm2(a) Before excitation by laser, (b) After 0.6 s excitation by laser (g) (i), (ii), (iii), (iv) mean signal of AuNRs, YO-PRO-1, transparent light and propidium iodide respectively. From Fig. (e), The apoptosis of the tumors can be observed. ............. . 59 3.7 Photothermolysis of the EMT-6 tumor cell triggered by PSS-AuNRs (N~30–40 clusters) under energy fluence 5.9 mJ/cm2(a) Before excitation by laser, (b) (c) (d) (e) (f). After 0.6 s, 360 s, 720 s, 1080 s, 1860 s excitation by laser (g) (i), (ii), (iii), (iv) mean signal of AuNRs, YO-PRO-1, transparent light and propidium iodide respectively. From Fig. (g). The apoptosis of the tumors can be observed .................................................... 60 3.8 Photothermolysis of the EMT-6 tumor cell triggered by PSS-AuNRs (N~2–15 clusters) under energy fluence 24.2 mJ/cm2 ..(a) Before excitation by laser, (b) (c) (d) (e) (f) After 0.6 s,210 s, 630 s, 900 s, 1230 s excitation by laser (g) (i), (ii), (iii), (iv) mean signal of AuNRs, YO-PRO-1, transparent light and propidium iodide respectively. From Fig. (g), the apoptosis of . v .

(10) the tumors can be observed. ................................................. 61 3.9 Photothermolysis of the EMT-6 tumor cell triggered by PSS-AuNRs (N~2–15 clusters) under energy fluence 5.9 mJ/cm2 ... (a) Before excitation by laser, (b) After 0.6 s excitation by laser (e) (i), (ii), (iii), (iv) mean signal of AuNRs, YO-PRO-1, transparent light and propidium iodide respectively. From Fig. (e), after observation for 1180 s, YO-PRO-1 and propidium iodide do not penetrate the comparatively leaky membranes. It figures that that the tumor cell is still alive instead of destruction by photothermolysis................................................................. 62 3.10 The situation of cancer cells to the power after photothermal therapy. ................................................................................ 62 3.11 Power test of non-treated mice ears (a) 1.2 W/cm2; (b) 1 W/cm2; (c) 0.8. W/cm2; (d) 0.6 W/cm2.. From these. results, the limited power density of 0.8 W/cm2 was observed obviously ............................................................................ 64 3.12 Control group (tumors only) (a)~ (f) are before induced tumors, and after induced for 5 days, 8 days, 11 days, 15 days, 20 days, respectively ................................................................ 65 3.13 Control group (tumors exposed by laser) (a)~ (f) are before induced tumors, and after induced for 5 days, 8 days, 11 days,15 days, 20 days, respectively.. Exposure with laser on. the 12 nd day, power density is 0.6 W/cm2, for 120 s.. From. this result, it can be observed that the laser was excluded as a factor, which destroys the solid tumors. ............................ 66 3.14 Control group (tumors with PSS-AuNRs) Fig. (a)~ Fig. (f) are . vi .

(11) after induced for 5 days, 8 days, 11 days, 15 days, 20 days, 24 days, respectively. 10 µg PSS-AuNRs were intratumorally injected into the solid tumor on the 11st day. From this result, it can be observed that the PSS-AuNRs was excluded as a cytotoxic factor. .................................................................... 66 3.15 Experimental group, the tumors were exposed with laser on the 12nd day, power density is 0.6 W/cm2, for 120 seconds. (a)~(g) are after induced for 5 days, 8 days, 11 days, 15 days, 20 days, 24 days, 28 days, respectively.After calculation for the tumors size, the growth curve was presented in Fig. 3.15. ...................................................................................................... 67 3.16 Growth Curve. The green-colored, blue-colored, black-colored and the red-colored symbol mean growth curve of tumors, tumors with laser, tumors with AuNRs and tumors with AuNRs under exposed by laser, respectively. ..................... 68 3.17 Histological sections of tumor tissues (a) Normal cells (b) EMT-6 (c) EMT-6 under exposure with 0.6 w/cm2 laser for 2 min (d) EMT-6 with PSS-AuNRs injected (e) 1 day after photothermal treatment (f) 8 day after photothermal treatment..... ...................................................................................................... 69 . . vii .

(12) Chapter 1 Introduction. According to the survey of the department of health, cancer has been the first among the top ten leading causes of death in the past 29 years in Taiwan. It is very urgently to develop effective and efficient therapy to the cancer. At present, most of the therapies destroy the cancer cells but meanwhile the normal cells, so it is a very significant issue to increase the viability of normal cells during the therapy. In in vitro experiments, there are excellent results that tumor cells were destructed by photothermal therapy of AuNRs. Photothermal therapy is good at localized destruction without killing too many normal cells, which are near the tumor cells. Taking this point as an advantage, we want to destroy the tumor cells with the photothermal therapy in in vivo experiments. In in vivo experiments, a great amount of heat that produced by the excitation of AuNRs destroys the tumor cells. Using the 808 nm as the excited source due to the poor absorption of the bio-organs. In order to ensure that the photothermal effect as the dominant cell-killing factor, higher biocompatible PSS-coated with AuNRs is . 1 .

(13) necessary, the PSS-coated excludes other possible influences towards cell viability.. 1.1 Properties of AuNRs Many physical properties of materials change as their size decreases, including the effect of statistical mechanics and quantum mechanics within. Nanotechnology can be regarded as a manipulation of these properties in atomic and molecular scale within the realm of traditional science, whereas traditional disciplines. can. be. re-interpreted. as. the. application. of. nanotechnology. Inter-related areas of research include physics, chemistry,. biology,. mechanical. engineering,. and. electrical. engineering. Furthermore, since nanotechnology is multi-directional and these areas of studies often overlap, physical chemistry, materials science, and biomedical engineering are also regarded as essential components of nanotechnology. Materials would show very different characteristics at nanometer scale than at macro scale, which makes some unique applications possible. For example, opaque materials become transparent (Copper); inert materials become catalysis (Platinum); stable . 2 .

(14) materials become inflammable (Aluminum); Liquid gold becomes red under nanometer scale; insulators become conductors (Silicon). The unique quantum and physical phenomena of materials under nanometer. scale. have. lead. to. the. many. branches. of. nanotechnology, along with fair attention to the applications of new materials.. 1.1.1 Surface plasmon resonance [1] When a metal nanoparticle is irradiated with electromagnetic radiation, if the frequency matches the material characteristic resonant frequency, all the ‘free’ electrons within the conduction band of the particle will undergo an in-phase oscillation with the frequency of the radiation. The oscillating electromagnetic field induces a polarization of the free conduction electrons with respect to the nanoparticle’s ionic metal core; the effect, in turn, establishes a restoring Coulomb force. In this manner, a dipolar oscillation of the electrons is created: a phenomenon generally known as surface plasmon resonance (Bohren and Huffman 1983), as shown in Fig. 1.1. Meanwhile, this SPR sets off, through dissipation, a strong absorption of radiation; the effect results in a broad absorption band centered in a certain optical absorption spectrum. . 3 .

(15) These pronounced absorptions are characteristics of metallic nanoparticles. Having said that, we will see that, long AuNRs, being strong absorbers of near infrared radiation, are the most prospective candidates as medical photothermal convertors.. Fig. 1.1, the schematic illustration of plasmon oscillation in an AuNR under light irradiation. The resonance of electrons across the particle can be induced by the electromagnetic field of the light. (Published material of Cheng-Lung Chen).. 1.1.2 Quantum confinement effect The quantum confinement effect can be observed once the diameter of the particle is of the same magnitude as the wavelength of the electron wave function. When materials are this small, their electronic and optical properties deviate substantially from those of bulk materials. A particle behaves as if it were free when the confining dimension is large compared to the wavelength of the particle. During this . 4 .

(16) state, the band gap remains at its original energy due to a continuous energy state. However, as the confining dimension decreases and reaches a certain limit, typically in nano scale, the energy spectrum turns to discrete. As a result, the band gap becomes size dependent. This ultimately results in a blue shift in optical illumination as the size of the particles decreases. Specifically, the effect describes the phenomenon resulting from electrons and electron holes being squeezed into a dimension that approaches a critical measurement, called the excition Bohr radius. In quantum mechanics view: The following equation shows the relationship between energy level and dimension spacing:. 𝜓!! ,!! ,!! =. 𝑛! 𝜋𝑦 8 𝑛! 𝜋𝑥 𝑛! 𝜋𝑧 sin( ) sin( ) sin( ) 𝐿! 𝐿! 𝐿! 𝐿! 𝐿! 𝐿!. 𝐸!! ,!! ,!!. . 𝑛! ħ! 𝜋 ! 𝑛! ! 𝑛! = [( ) + ( )! + ( )! ] 2𝑚 𝐿! 𝐿! 𝐿!. 5 .

(17) 1.2 Introduction of cancer Cancer claims one of the top ten causes of death in Taiwan in the recent years. Cancer is characterized by the lack of apparent symptoms in its early stages, and that treatments for cancer can be difficult and ineffective. All cancers begin in cells. Normal body cells undergo controlled growth and divisions, expressed by their genetic materials, to form functional tissues and organs. The different types of cells in the body, including intestinal mucous cells, epithelial cells of skin, and hematopoietic precursor cells in bone marrow, continuously proliferate, divide, grow, die and replaced as they become old or damaged. However, loss of normal growth control could happen when mutations occur in DNA due to genetic problems or environmental risk factors such as infections, radiations, and carcinogens. In fact, mutations occur regularly in normal controlled cell division but these mutated cells quickly undergo apoptosis before turning into tumor cells. Once the bodies lose control of cell division, mutated cells could proliferate indefinitely, and directly invade neighbouring tissues or spread to other organs through the lymph system and vascular networks. . 6 .

(18) Moreover, tumor cells continuous to grow uncontrollably when they reach distal organs, where they damage the tissues and functions of the organs and lead to complications.. 1.2.1 Differences between cancer and normal cells Tumor cells stems from normal cells, however several major differences exist between the two: 1) Cell Structure: Under normal condition, regular cells from the same tissue have relatively the same size and shape, however, tumor cells originate from a tissue are much bigger than a normal cell of the same tissue because the nucleus of a tumor cell is bigger and of a different shape. In addition, cases of giant nuclei, multiple nuclei, or distorted nuclei are also possible. 2) Cell Growth: The growth of tumor cells has two characteristics a) Autonomous growth: Under normal cell growth, divisions cease when the proliferating cells come in contact with another cell. On the other hand, tumor cells continue to proliferate and divide abnormally at a higher growth rate without control, thereby growing on top of each other or erode into the surrounding tissue. b) Metastasis: A tumor can be non-cancerous or cancerous, differentiated by cancerous cells' ability to metastasize and erode . 7 .

(19) into the surrounding structure, because the motility of normal cells are limited by the adhesive force existing between them. The ability of cancerous cells to migrate to nearby and distal locations is due to the loss of adhesive force between cells as a result of structural change on cell surfaces, which cause the cancerous cells to erode surrounding tissue and travel to different parts of the body through blood and lymphatic vessels. 3) Cell Metabolism: Metabolism is necessary to keep a cell alive and functional. However, a great difference in metabolic rate is found between a normal and tumor cell, especially in protein and nucleic acid synthesis. Protein synthesis is increased in tumor cell, while the breakdown of protein is slowed, leading to an imbalance between the rate of protein synthesis and degradation. 4) Malignity and Heredity: Tumor cells have higher malignity and growth rate, and most are capable of metastasis since early stages of cancers, hence cancers are often difficult to cure. Furthermore, tumor cells can pass down their ability of autonomous growth to the next generation of tumor cells, producing proliferating cells of the same malignant characteristics.. . 8 .

(20) 1.2.2 Metastasis of cancer cells Traditionally, metastasis of cancerous cells is thought to occur only during later stages of tumor growth. However, the recent research suggests that most of tumor cells are already capable of metastasis during the early stages.. In other words, migration to. distal sites could happen during early stages of tumor formation. Many recent findings indicate that non-cancerous and cancerous tumor cells do not share exactly the same genetic materials or characteristics. In general, there are two routes of metastasis: a) Lymphatic spread is one route of metastasis, where the tumor cells proliferate at regional lymph nodes and the invasion eventually becomes clinically detectable. Towards the later stages of cancers, some of the tumor cells at the lymph nodes may be cancerous cells, which then metastasize to other parts of the body through the lymphatic system, leading to tumor formations at distal sites such as the head and neck. b) Haematogenous spread is another route of metastasis, and it occurs during the early stages of cancers through blood vessels. Tumor cells may reach any part of the body, even bone marrows, . 9 .

(21) but these tumor cells have short life span and do not have the capacity for rapid proliferation.. However, a small number of the. metastasized tumor cells could still have rapid growth rate, therefore a second metastasis is possible. An example of cancer as a result of haematogenous spread is breast cancer. Beside metastasizing into neighbouring tissue, cancerous cells at the site of origin are even more malignant and have began metastasizing much earlier through blood capillary network or lymphatic vessels to regional lymph nodes or other organs. However, metastasis requires several steps to complete, including: 1) Cancerous cells are nourished by self-generating autocrine factors as well as the paracrine factors produced by surrounding tissues. 2) When the diameter of a cancerous cell has increased to 1-2 millimeters, it will become dependent on the formation of capillary networks around the tumor for nourishment and growth. At this point, some of the tumor cell adhesion molecules would undergo negative. regulation,. thereby. encouraging. cell. fluidity. and. detachment from the site of origin into surrounding blood circulations. . 10 .

(22) 3) Cancerous cells metastasize through blood capillaries into other body parts including, lungs, liver, and bones. There are receptors on the surface of the cancerous cells that react with the paracrine signals from various organs, providing these cancerous cells with sufficient nutrients for continuous growth. 4) Therefore, most clinical cases of metastasis are of multiple metastatic lesions, where the cancerous cells are heterogeneous in gene expressions. The trend for future research in cancer treatment should then be finding out the specific genetic properties of the metastasising cells and formulating a targeted therapy.. 1.3 Cancer therapies The choice of therapy depends upon the type and location of the tumor and the stage of the disease. The types of cancer treatment commonly applied today are listed as follow: a) Surgery:. Tumors, and potential areas that could be affected or. metastasized, can be directly removed by surgery. In general, 35% of cancers can be cured by surgery, and other types of treatment cannot cure these cancers. Only one third of the cancer cases can . 11 .

(23) be cured by surgery because the effectiveness of surgery is only limited to patients with cancers of earlier stages, especially those of intestinal, colon, kidney, breast, and ovarian cancers, where 80% of the disease could be cured.. However, when the cancer has. spread to lymph nodes prior to surgery, the chance of complete surgical excision is only as low as 20%.. Furthermore, complete. cure of cancers by surgery is impossible, with a few exceptions, when cancerous cells have spread to the rest of the body. Although surgery may be inappropriate in later stages of the disease, it could be used to control symptoms and prolong the lives of patients. b) Chemotherapy: Chemotherapy is the treatment of cancer using anticancer drugs. Normally, cells are under controlled divisions and growth, however, cancer cells are rapidly dividing and incapable of being controlled. Chemotherapy affects the abnormal cells via drugs that damage the rapidly dividing cells. The delivery of anticancer drugs may be through oral method or injections via skin, muscles, or veins, where injection through veins is the most common. However, bone marrow repression is a common side effect of chemotherapy that lowers the manufacturing of haemoglobins, leading to hypotension, loss of blood, and infections . 12 .

(24) and fevers due to reduction in white blood cells. Other side effects include, hair loss, abdominal problems (such as oral mucosa tearing off, diarrhea, constipation), nausea, poor appetite, and fatigue. Moreover, some drugs may also cause nervous system problems, including limb numbness and fatigue. In general, physicians would give precautions or post-treatments to possible side effects. However, some side effects may cease after chemotherapy, such as hair loss and abdominal problems. c) Radiation Therapy: High dosage of radiation could kill cells as well as prevent their divisions and growth. Since tumor cells grow and divide much rapidly than surrounding normal cells, radiation therapy is effective against many types of cancer. However, the cell cycle of normal cells would also be affected. In order to avoid disruption in normal cell cycle, targeted areas and radiation dosage are highly controlled.. Multisegmented therapy is also important.. Although radiation therapy accompanies disturbing side effects, but these side effects are not serious and can be controlled by medicines or diets. Some side effects may last for a longer time, but will eventually cease in a few weeks after the completion of therapy. . 13 .

(25) d) Targeted Therapy:. Targeted therapy uses drugs to target. specific molecules on the surface of tumor cells and those in the tumor cell growth signalling pathway in order to suppress cancerous cell growth and progression. This type of therapy may also induce apoptosis to the tumor cell by disrupting the development of blood vessels around the tumor. Targeting drugs may work differently: 1) Most drugs interfere with cell signalling pathways by blocking signals that govern tumor growth and cancerous cell activities, causing tumor progression to cease due to inability to response to external stimuli. 2) Other drugs target and block the growth of blood vessels to tumors. Tumors require a blood supply during growth to receive nutrients, which means treatments that interfere with blood vessels will induce apoptosis to tumors. 3) Some targeted therapies modify the function of proteins that regulate gene expression and other cellular functions by suppressing tumors' ability to produce proteins. 4) Some drugs dissolve intercellular substances to restrict further . 14 .

(26) growth of tumor cells. However, this method has no apparent effectiveness so far and is still under investigation. e) Immunotherapy: Immunotherapy is also known as biotherapy, which stimulates the immune system to attack tumor cells that are responsible for the cancer. Immunotherapy can be broken down into monoclonal antibody therapy (adaptive immunotherapy), immunization of cancer vaccine, and non-specific immunotherapy and adjuvants. 1) Monoclonal antibody therapy: It is the most commonly immunotherapy used today, where artificial antibodies are raised, rather than self-produced, against specific antigens present on the surfaces of tumors, thereby recognized as passive immunity. In the last ten years, the U.S. Food and Drug Administration (FDA) have approved many therapeutic monoclonal antibodies for use in humans. 2) Cancer vaccine:. The vaccine may composed of cancer cells,. parts of cells, or pure antigens to increase the immune response against cancer cells that are already in the body. In addition, vaccines are often combined with other substances of cells, known. . 15 .

(27) as adjuvants, to enhance the immune response even further. Cancer vaccine is considered active immunity since it induce patient's own immune response against cancer antigens, and it is also very specific, though often requires more than one adjuvant. 3)Non-specific immunotherapy and other adjuvants: Interleukin is one of the cytokines body produces, in which interleukin-2 (aldesleukin; brand name: Proleukin) has been approved by FDA for clinical use against metastatic cutaneous melanoma and kidney cancer. f) Photodynamic Therapy: This therapy activates photosensitizing agents using low power laser beans. The energy level of photosensitizing agents reach a high and unstable state under stimulation, and when drop down and return to their stable state, energy is released along with free radicals and oxygen atoms. The free radicals and oxygen atoms oxidize and harm the cancer cells. The approved photosensitizing agent for retinal neovascularisation photodynamic therapy is BMP-MA (brand name: Visudyne), which is highly lipophlic and could combine with LDL. The amount of LDL receptors in newly formed microvascular networks is ten time of. . 16 .

(28) that found in mature blood vessels, which means BMP-MA can selectively gather on the abnormal neovascular networks and harm them under light stimulation by inducing platelets to form plaque and obstruct the neovascular networks.. . 17 .

(29) Chapter 2 Experimental details 2.1 Chemicals 1.. Cethyltrimethylammonium bromide，for molecular biology， CTAB: N(C16H33)(CH3)3Br，99% for molecular biology， SIGMA 十六烷基-三甲基-溴化銨. 2.. Silver nitrate: AgNO3，99.95% for analysis，ACROS (硝酸銀). 3.. L-Ascorbic acid: C6H7NaO6，99%，ACROS (維他命 C). 4.. Sodium borohydride: NaBH4，ACROS (氫硼化鈉). 5.. Hydrogen tetrachloroaurate (III) hydrate: HAuCl4 .xH2O， ACROS (四氯氫化金). 6.. Poly(sodium 4-styrenesulfonate)，PSS: C8H7NaO3S， 30wt.% solution in water， MW7000，ALORICH 聚對苯乙烯磺酸鈉. 7.. Sodium hydroxide: NaOH，99%，SIGMA (氫氧化鈉). 8.. Sodium chloride: NaCl，ACROS (氯化鈉). 9.. Hank's balanced salt solution，HBSS: (GIBCO 漢克平衡鹽緩衝液). 10.. Dulbecco’s Modified Eagle Medium/Nutrient Nixture F-12， DMEM/F12(1:1): with 15 mMHEPES、L-Glutamine，GIBCO (細胞培養液). . 18 .

(30) 11.. TRYPSIN-EDTA: 5gr/l TRYPSIN、2gr/l (5.3mM) EDTA Na4 in SALINE，invitrogen (胰蛋白酶). 12.. (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT)，invitrogen. 13.. Dimethyl sulfoxide, DMSO: (CH3)2SO，99.5%，SIGMA (二甲基亞碸). 14.. Penicillin Streptomycin，Pen Strep: 10000 Units/mL Penicillin+10000ug/mL Streptomycin，GIBCO (盤林西林). 15.. BIOMYC-3: concentrate x 100，invitrogen (抗黴素). 16.. Fetal Bovine Serum，HyClone (血清). . 19 .

(31) 2.2 Instruments 1. UV-visible spectroscopy Model： SPECTRONIC GENESYS-8 2. Transmission Electron Microscopy，TEM Model：JEM-2100F 3. Zeta Potential Model：Malvern Zetasizer Nano ZS90 4. Inverted Confocal Microscopy Model：Zeiss LSM 510 META NLO DuoScan 5. Fluorescence Stereomicroscope Model：Leica MZ16 FA 6. Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) Model: Thermo Xseries II. . 20 .

(32) 2.3 Preparation of AuNRs[2, 3]. 2.3.1 Synthesis of AuNRs There are two methods to prepare AuNRs. (A) One pot synthesis method (B) Seed-mediated growth method Procedure (A). One pot synthesis method. I. Preparation of growth solution: 1. 50 mL of 0.2M CTAB aqueous solutions. Take 3.645 g of CTAB powder and dissolve in 50 mL of DI water (under sonication). 2. 100 mL of 20mM AgNO3 aqueous solution. Take 0.34 g of AgNO3 powder and dissolve in 100mL of DI water. 3. 20 mL of 0.1M L-ascorbic acid aqueous solutions. Take 0.396 g of L-ascorbic acid powder and dissolve in 20mL of DI water. 4. 100 mL of 4mM NaBH4 aqueous solution. Take 0.015 g of NaBH4 powder and dissolve in 100 mL of ice-cold water.. . 21 .

(33) II. Growth of gold nanorods: 1. Take 250 µL of 0.1M HAuCl4 aqueous solutions and mix with 25 mL of 0.2M CTAB aqueous solutions. 2. Add 250 µL of 20mM of AgNO3 aqueous solution into the above solution. 3. Add 280 µL of 0.1M L-ascorbic acid aqueous solutions into the above solution. The application must be done slowly, with the solution transparent at the end. 4. Finally 38 µL of 4mM NaBH4 aqueous solution added to the solution. A color should be observable within 10 minutes. 5. Gently stir solution for 3 hours till completion. 6. Centrifuge the solution at 14000 rpm for 20 minutes and re-disperse in DI water. (B). Seed-mediated growth method. I. Preparation of growth solution: 1. 50 mL of 0.001 M HAuCl4 stock aqueous solution. From previously prepared 0.1 M HAuCl4 aqueous solution, take 0.5 mL and dilute to 50 mL. 2. 50 mL of 0.2M CTAB aqueous solutions. Take 3.645 g of CTAB powder and dissolve in 50 mL of DI water (under sonication). . 22 .

(34) 3. 10 mL of 0.004M AgNO3 aqueous solutions. Take 0.068 g of AgNO3 powder and dissolve in 10mL of DI water, then take 1 mL from this solution then dilute to 10 mL. 4. 10 mL of 0.0788M L-ascorbic acid aqueous solutions. Take 0.156 g of L-ascorbic acid powder and dissolve in 10mL of DI water. 5. 10 mL of 0.01M NaBH4 aqueous solutions Take 0.0378 g of NaBH4 powder and dissolve in 10 mL of ice-cold water, then take 1mL from this solution then dilute to 10 mL ice-cold water II. Growth of gold seeds 1. Prepare 0.0005 M of HAuCl4 by taking 1 mL of 0.001 M and dilute to 2 mL. 2. Mix 2 mL CTAB aqueous solution with the above solution, and then stir thoroughly. 3. Add 240µL of ice-cold NaBH4 to mixed solution while stirring, stir vigorously for a further 2 minutes. The solution should appear brownish-yellow. 4. Heat to 40~45°C with gentle stirring for 15 minutes. This should remove excess NaBH4.. . 23 .

(35) III. Growth of AuNRs 1. Take 15 mL of 0.001M HAuCl4 aqueous solutions and mix with 15 mL of 0.2M CTAB aqueous solutions. 2. Add 800 µL of 0.004 M of AgNO3 aqueous solution into the above solution. 3. Add 160 µL of 0.0788 M L-ascorbic acid aqueous solution into the above solution. The application must be done slowly, with the solution transparent at the end. 4. Finally 29 µL of gold seed is quickly added to the solution. A color should be observable within 10 minutes. 5. Gently stir solution for 3 hours till completion. 6. Centrifuge the solution at 14000 rpm for 5 minutes and re-disperse in DI water. Finally choose method (A) as the primary synthesis, since this method makes much more gold nanorods during the same time.. 2.3.2 Surface modification with polystyrenesulfonate 1. 50 mL of 1 mM NaCl aqueous solution Take 2.92 mg of NaCl powder and dissolve in 50mL of DI water. 2. Add 1.4 mL of PSS (poly (sodium 4-styrenesulfonate)) aqueous solution into the above solution. . 24 .

(36) Preparation of PSS-AuNRs 1. 15 mL of CTAB-AuNRs aqueous solution was centrifuged at 14000 rpm for 6 minutes and re-disperse in DI water. 2. Add 15 mL PSS- aqueous solution and gently stir solution for ~24 hours till completion. 3. The above procedure was repeated at least three times. 4. Centrifuge the solution at 10000 rpm for 20 minutes and re-disperse in DI water.. Fig. 2.1 Procedure of one-pot synthesis of AuNRs. . 25 .

(37) 2.3.3. Characterizations of AuNRs. 2.3.3.1 UV-visible spectroscopy Choosing 800 nm as the excitation source is good for photothermal therapy, since the poor absorption of bio-organism at near-infrared light.. Fig. 2.2 Absorption of bio-organism An obvious difference between certain compounds is their color. To understand why some compounds are colored and others are not, and determine the relationship of conjugation to color, making accurate measurements of light absorption at different wavelengths from ultraviolet to visible light of the spectrum is necessary.. . 26 .

(38) Fig. 2.3 Visible spectrum Molecules. containing. π-electrons. or. non-bonding. electrons. (n-electrons) can absorb the energy in the form of ultraviolet or visible light to excite these electrons to higher anti-bonding molecular orbitals. The more easily excited the electrons, the longer the wavelength of light it can absorb. The Beer-Lambert law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length.. A = log!". I! 1 = log!" = K𝑙c I! T. where 𝐴: Absorbance, 𝐼! : Intensity of incident light, 𝐼! : Transmitted intensity, 𝑇: Transmittance,𝐾: Constant of the molar absorptivity or extinction coefficient, 𝑙 : Pathlength through the sample, 𝑐 : Concentration of the absorbing species. . 27 .

(39) Preparation of sample Take 1 mL of CTAB-AuNRs and PSS-AuNRs respectively, and place the solution in cuvette.. 2.3.3.2 Transmission electron microscope (TEM) TEM is capable of imaging at a significantly higher resolution than light microscopes, due to the de Broglie wavelength of electrons. This enables the instrument's user to examine fine detail. It’s a major analysis method in both physical and biological sciences. Resolution. Magnification. Eyes. 0.2 mm. 1:1. Optical Microscopy. 0.2 µm. 1000:1. SEM. 3.5~10 nm. 2000:1. TEM. 0.2 nm. 1000000:1. Fig. 2.4 Comparisons of microscope An electron source emits the electrons that travel through vacuum in the column of the microscope. The TEM uses electromagnetic lenses to focus the electrons into a very thin beam instead of glass lenses focusing the light. The electron beam then travels through the specimen. Depending on the density of the material present,. . 28 .

(40) some of the electrons are scattered and disappear from the beam. At the bottom of the microscope the un-scattered electrons hit a fluorescent screen, which gives rise to a "shadow image" of the specimen with its different parts displayed in varied darkness according to their density. The image can be studied directly by the operator or a CCD.. Fig. 2.5 The structure of TEM. 2.3.3.3 Zeta potential measurement It is a measurement of the magnitude of the electrostatic or charge repulsion or attraction between particles; the amount of charge on the particle surface is an important particle characteristic. . 29 .

(41) because it determines many of the properties of the suspension or emulsion. The results of Zeta potential can distinguish whether CTAB-AuNRs is transformed into PSS-AuNRs on the surface. When an electric field is applied across an electrolyte, charged particles suspended in the electrolyte are attracted towards the electrode of opposite charge. Viscous forces acting on the particles tend to oppose this movement. When equilibrium is reached between these two opposing forces, the particles move with constant velocity. The velocity of the particle is dependent on the following factors: a. Strength of electric field or voltage gradient. b. The Dielectric constant of the medium. c. The Viscosity of the medium. d. The Zeta potential. The velocity of a particle in an electric field is commonly referred to as its electrophoretic mobility. With this knowledge the zeta potential obtained of the particle by application of the Henry equation.. . 30 .

(42) the Henry’s equation is :. 𝑈! =. 2𝜀𝑧𝑓(𝑘𝑎) 3𝜂. 𝑧: Zeta potential, 𝑈! : Electrophoretic mobility, 𝜀: Dielectric constant, 𝜂: Viscosity, 𝑓(𝑘𝑎): Henry’s function. Fig. 2.6 Schematic of zeta potential measurement Preparation of the sample: Take 10 µL of O.D.=1 CTAB- and PSS- gold nanorod respectively, and dilute to 1 mL DI water, placing the solution in zeta potential cell.. Fig. 2.7 Zeta potential cell. . 31 .

(43) 2.3.3.4 Inductively coupled plasma mass spectroscopy (ICP-MS) [4] ICP-MS is a method of mass spectroscopy that is highly sensitive and capable of the determination of a range of metals and several non-metals at concentrations below one part in 1012. It is based on coupling together inductively coupled plasma as a method of ionization with a mass spectrometer as a method of separating and detecting the ions. ICP-MS comprises five basic steps: 1. Generating an aerosol of the sample 2. Ionizing sample in the ICP source 3. Extracting ions in the sampling interface 4. Separating ions by mass 5. Detecting ions, calculating the concentration. Fig. 2.8 Schematic of ICP-MS measurement(ref) . 32 .

(44) Preparation of samples: 1 mL of PSS-AuNRs aqueous solution was centrifuged at 14000 rpm for 25 minutes and re-disperse in aqua regia. Dilute the aqueous solution to 10 mL in DI water. Take the above aqueous solution dilute 1000-fold as the sample 1, and then take 1 mL of sample 1 dilute to 10 mL DI water, as sample 2.. Fig. 2.9 Preparation process of PSS-AuNRs with two different concentrations.. 2.4 In Vitro Experiments. 2.4.1 Cell line EMT-6 is breast cancer cell from BALB/cCrgl, ATCC® Number: CRL-2755™ . 33 .

(45) 2.4.2 Cell culture Cell culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. Procedure 1.. Remove and discard culture medium.. 2.. Briefly rinse the cell layer with HBSS buffer solution to remove all traces of serum that contains trypsin inhibitor.. 3.. Add 1 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes). To avoid clumping do not agitate the cells by hitting or shakingthe flask while waiting for the cells to detach. Cells that are difficult to detachmay be placed at 37°C, 5% CO2 to facilitate dispersal.. 4.. Add 10 mL of complete growth medium and aspirate cells by gently pipetting.. 5.. Add appropriate aliquots of the cell suspension to new culture vessels. Incubate cultures at 37°C, 5% CO2.. . 34 .

(46) 2.4.3 Cell viability assay (MTT Assay) [5] Colorimetric assay is a popular method in quantifying cell survival percentage. In principle, the absorbance of formazan which. was. produced. from. the. mitochondrial. oxidation. of. 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) in living cells is directly proportional to the number of living cells.. Fig. 2.10 Molecular of structure (MTT) Procedure: 1. 6000 EMT-6 cells were planted into each well of 96-well plates with 300 µL medium and incubated at 37°C under a 5% CO2 atmosphere for 24 hours. 2. These cells were further incubated for 12, 24 and 48 hours with CTAB-AuNRs. and. PSS-AuNRs. respectively. at. varying. concentrations. 3. Treated with a freshly prepared 12 mM MTT solution (100µL) and incubated for additional 3 hours at 37°C under a 5% CO2 . 35 .

(47) atmosphere. 4. MTT solutions were removed and 50µL DMSO was added to each well. 5. The 96-well plates were left for half an hours in the dark, and then assayed with a spectrometer, fixing the absorbance at 550 nm. 6. The obtained cell viability was expressed as a percentage relative to cells incubated with medium only.. 2.4.4 In vitro photothermal therapy [6] Electromagnetic near infrared light are used to destroy the cancer cells. Making it absorb light energy of certain range excites the photosensitive material, AuNRs. The wavelength of light decides the energy to be supplied to the material for making it excited. The advantage of this therapy is that NIR is less powerful than the light energy that is used in photodynamic therapy. This feature ensures the safety of the normal cells and tissues that are near the target tumor. Since the good ability of surface plasmon resonance of AuNRs, it’s effective in absorbing the light energy and producing a great amount of heat to destroy the cancer cells.. . 36 .

(48) 2.4.4.1 Introduction to fluorescent dyes Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Dyes are frequently used in biology to highlight structures in biological tissues for viewing, often with the aid of different microscopes. YO-PRO-1 and PI (Propidium iodide) are used to observe the structures of cancer cells during the process this experiment. YO-PRO-1. stain. selectively. passes. through. the. plasma. membranes of apoptotic cells and labels them with moderate green fluorescence. Necrotic cells are stained red-fluorescent with propidium iodide.. . 37 .

(49) (i). YO-PRO-1: Molecular formula is C24H29I2N3O. Fig. 2.11 Absorption and fluorescence emission spectra of YO-PRO-1 bound to DNA.. . 38 .

(50) (ii). Propidium Iodide: Molecular formula is C27H34I2N4. Fig. 2.12 Absorption and fluorescence emission spectra of propidium iodide bound to DNA.. . 39 .

(51) Fig. 2.13 Fluorescent images of destroyed cell. (a) Transparent pattern. (b) After the excitation, the emission signal (green) by YO-PRO-1 was detected. (c) After the excitation, the emission signal (red) by PI was detected. (d) Emerge signals of (a), (b), (c). 2.4.4.2 Confocal microscope and two-photon Iuminescence imaging (Zeiss LSM 510 META NLO DuoScan) I. Confocal Microscope: Improvement of fluorescence microscope image is the fundamental function of confocal microscope. Compare with the conventional fluorescence microscope, confocal microscope uses laser instead of mercury lamp as its light source. Conducted by the scanner mirrors, the laser beam excites. . 40 .

(52) specimens and the detector detects the emission fluorescence signal of the specimen point-by-point and line-by-line. The spatial pinhole eliminates out-of-focus light in specimens that are thicker than. the. focal. plane.. It. enables. the. reconstruction. three-dimensional structures from the obtained images.. Fig. 2.14 The difference between conventional and confocal microscope. The image indicates that the confocal image has better resolutions than the conventional image.. . 41 . of.

(53) Fig. 2.15 Beam path of the excitation and emission light in a confocal laser-scanning microscope. II.. Two-photon Iuminescence image. Two-photon excitation is based on the idea that two photons of comparably lower energy than needed for one photon excitation can also excite a fluorophore in one quantum event. Each photon carries approximately half the energy necessary to excite the molecule. An excitation results in the subsequent emission of a fluorescence photon, typically at a higher energy than either of the two excitatory photons. The probability of the near-simultaneous absorption of two photons is extremely low. Therefore a high flux of. . 42 .

(54) excitation photons is typically required, usually a femtosecond laser. The most commonly used fluorophores have excitation spectra in the 400–500 nm range, whereas the laser used to excite the two-photon fluorescence lies in the ~700–1000 nm (infrared) range. In this case, the surface plasmons absorb two near infrared photons simultaneously; it will absorb enough energy to transit into the excited state .The surface plasmons will then emit a single photon with wavelength that typically in the visible spectrum. Because two photons are absorbed during the excitation of the surface plasmons, the probability for fluorescent emission from the surface plasmons increases quadratically with the excitation intensity.. Fig. 2.16 Differences between single photon and two-photon excitation.. . 43 .

(55) Fig. 2.17 Two-Photon Iuminescence Images of gold nanorods. (a) Only the emission signal of AuNRs. (b) Signal by merge (a) and transparent light.. 2.5 In Vivo Experiments. 2.5.1 Animal model [7, 8] Model: Ears of female Balb/c with ages 4 to 6 weeks. All animals are from BioLASCO Taiwan Co., Ltd There are several advantages for setting the ears of mice as model 1. This model provides much more accurate for calculating the volume of the tumor, as comparing with the rear flank model of the mice.. . 44 .

(56) 2. Due to the thickness of ear is very thin, there is almost no limited of the laser depth. 3. It’s easier to observe the phenomenon of angiogenesis.. Fig. 2.18 Schematic of the ear structure. Fig. 2.19 Image of the histologic section of the mice ear . 45 .

(57) 2.5.2 Anesthesia A. Trichloroacetic acid (TCA) B. Ether. 2.5.3 In vivo photothermal therapy [9,10]. 2.5.3.1 Experimental processes I.. Induce solid tumors. Prepare the suspended cancer cells ~106 in 30 µL medium, and inject it subcutaneously into the right and left ear of the female mice Balb/c, the mice were anesthetized with TCA. II. Inject the PSS-AuNRs solutions into the solid tumors When the tumors grew to approximately 30 mm3, PSS-AuNRs (10 µg in terms of gold nanorods amount), which were suspended in 10 µL DI water, were directly injected into the solid tumors of ears. III. Photothermal therapy After the injection of PSS-AuNRs for 22 hours, the mice’s ears were exposure under the 808 nm laser, power density 0.6 W/cm2 for 120 seconds. IV. Observation of solid tumors size Take photo by stereomicroscope and then import in software “Image J”. Obtain the tumor size (volume) by calculating the pixels. . 46 .

(58) Fig. 2.20 Set-up of the in vivo photothermal therapy.. Fig. 2.21 Schematic of the in vivo photothermal therapy.. . 47 .

(59) 2.5.3.2 Microscope system Observing the solid tumor by a stereomicroscope, Leica MZ16FA, The Leica MZ16 FA fluorescence stereomicroscope is the fluorescence stereomicroscope with greatest zoom capability (16:1), highest resolution (840 Lp/mm), highest magnification (115x with standard optics), a patented illumination/filter system for the most intense fluorescence on jet-black backgrounds, and a high-performance. transmitted-light. base. for. excellent. relief. contrast.. 2.5.3.3 Estimation of tumor size The calculation of solid tumor size:. 𝑇𝑢𝑚𝑜𝑟 𝑣𝑜𝑙𝑢𝑚𝑒 𝑚𝑚! = 𝑎𝑟𝑒𝑎(𝑚𝑚! )×𝑐(𝑚𝑚)×. 𝜋 6. where c is the thickness of solid tumor. The area was measured by importing the graph, which took by Leica MZ16FA, into software “Image J”. And the thickness was measured by vernier, take average for five times measurement.. . 48 .

(60) 2.5.3.4 Histological sections [11] For fixation, place the whole specimen into Bouin for 24 to 72 hours. Centrifuge the Bouin and re-disperse with 70 % alcohol for couple times,. in. order. to. remove. the. yellow-colored. with. 2,4,6-Trinitrophenol. After the above procedure, dehydrate it by 80%, 85%, 90%, 95%, 100% alcohol in sequence. Paraffin-embedded after immersing the specimen in the toluene, then do the microtome cutting to the specimen with 5~6 µm thickness. Finally staining the specimen by the method called Gomori rapid one step trichome, and the last step is coverslipping with Rapid Mounting Media (Merk).. . 49 .

(61) Chapter 3 Results and discussion 3.1 Characterizations of AuNRs The AuNRs were prepared by the one pot synthesis method using. hexadecyltrimethylammonium. surfactants,. CTAB–AuNRs.. bromide. However,. (CTAB). CTAB. has. as been. demonstrated to be a problem in application due to its cytotoxicity [Langmuir 22, 2 (2006)].. In order to make AuNRs suitable for for. clinical purposes, additional surface modifications are necessary. Here, polystyrenesulfonate sodium salt (PSS, 70 kDa) was, therefore, chosen as a peptizing agent and surfactant for the efficient removal of CTAB from AuNRs suspensions. and (b) show the TEM images of PSS-AuNRs.. Fig. 3.1(a). The longitudinal. plasmon bands of CTAB-coated and PSS-coated AuNRs are located near 800 nm (Fig. 3.2); the produced water-soluble PSS–AuNRs are extremely stable at room temperature for several months.. In order to figure out whether replacing the CTAB-coated. (positive charge) with PSS-coated (negative charge) AuNRs, the zeta-potential measurements of CTAB-coated and PSS-coated AuNRs were also performed. . 50 .

(62) Fig. 3.4(a) and (b) show the results of zeta potential of CTAB- and PSS-coated AuNRs, respectively.. Fig. 3.1 (a) TEM image of PSS-AuNRs. (b) High resolution TEM image a single AuNR. Fig. 3.2 Absorption PSS-AuNRs.. . spectroscopy. 51 . of. CTAB-AuNRs. and.

(63) Fig. 3.3(a) The zeta potential of CTAB-AuNRs is 45.2±13.8 mV.. . 52 .

(64) Fig. 3.3(b) The zeta potential of PSS-AuNRs is -39.5±16.3 mV.. . 53 .

(65) In addition, aliquots of AuNRs were also subjected to inductively coupled plasma optical mass spectroscopy (ICP-MS) to correlate the optical density (O.D.) with particle quantity.. Comparison. against a calibration line gave a gold content of ~20 µg/mL at O.D. = 1.. Fig. 3.5(a) and (b) show the calibration data and measured. data respectively. And Fig. 3.5(c) shows the results of ICP-MS analysis of PSS-AuNRs.. Fig. 3.4(a) Calibration data of Au.. . 54 .

(66) Fig. 3.4(b) Measured data of PSS-AuNRs.. Fig. 3.4(c) The results of ICP-MS measurement. The blue points are the standard curve of calibration, and the red points are the results of the analysis.. . 55 .

(67) 3.2 In vitro cancer cell photothermolysis mediated by AuNRs Using a standard colorimetric cell viability assay (MTT assay), the cytotoxicity of the CTAB-AuNR and PSS-AuNRs were evaluated against EMT-6 breast tumor cells, as shown in Fig. 3.5. The assay evidently verified that CTAB-AuNRs presented obviously cytotoxicity after incubation with the cells for 12, 24 and 48 h.. In comparison, the viability of cells with PSS-AuNRs, was. much higher than CTAB-AuNRs, confirming its biocompatibility with EMT-6 cells, and can thus be used in further studies pertaining to in vitro/in vivo imaging and photothermal therapeutics.. . 56 .

(68) Fig. 3.5 Cell viability of EMT-6 cells exposed to CTAB- and PSS-coated AuNRs after (a) 12 h, (b) 24 h and (c) 48 h incubation, respectively. The viability of the cells was normalized with respect to a media-only control.. By using the inverted confocal microscope, LSM 510 META, the destruction of tumor cells triggered by PSS-AuNRs assisted with two photon laser excitation can be observed in situ real-time.. The. cell destruction process can be dynamic monitoring by using the fluorescent dyes, YO-PRO-1 (green coloring) and propidium iodide (red coloring).. As the cell membrane becomes slightly permeable,. it will enable YOPRO-1 but not propidium iodide to enter the cytoplasm. However, upon cell death, propidium iodide can . 57 .

(69) penetrate the comparatively leaky membranes. Neither of two dyes can penetrate viable cells.. To realize the influence of different. power density and different quantity of PSS-AuNRs on the destruction of cancer cells, two different energy fluences and quantity of PSS-AuNRs were applied to living cells in sequence. The cell mortality was observed at energy fluence of 93 mJ/cm2. Results of the cells with AuNRs (N~30–40 clusters), under excitation at energy fluences of 24.2 and 5.9 mJ/cm2, are shown in the series of images of Fig. 3.6 and 3.7, respectively.. Additionally,. results of the cells with fewer AuNRs (N~2–15 clusters) under excitation at energy fluences of 24.2 and 5.9 mJ/cm2, are shown in the series of images in Fig. 3.8 and 3.9.. . 58 .

(70) Fig. 3.6 Photothermolysis of the EMT-6 tumor cell triggered by PSS-AuNRs (N~30–40 clusters) under energy fluence 24.2 mJ/cm2. (a) Before excitation by laser, (b) After 0.6 s excitation by laser (g) (i), (ii), (iii), (iv) mean signal of AuNRs, YO-PRO-1, transparent light and propidium iodide respectively. From Fig. (e), The apoptosis of the tumors can be observed.. . 59 .

(71) Fig. 3.7 Photothermolysis of the EMT-6 tumor cell triggered by PSS-AuNRs (N~30–40 clusters) under energy fluence 5.9 mJ/cm2. (a) Before excitation by laser, (b) (c) (d) (e) (f). After 0.6 s, 360 s, 720 s, 1080 s, 1860 s excitation by laser (g) (i), (ii), (iii), (iv) mean signal of AuNRs, YO-PRO-1, transparent light and propidium iodide respectively. From Fig. (g). The apoptosis of the tumors can be observed.. . 60 .

(72) Fig. 3.8 Photothermolysis of the EMT-6 tumor cell triggered by PSS-AuNRs (N~2–15 clusters) under energy fluence 24.2 mJ/cm2. (a) Before excitation by laser, (b) (c) (d) (e) (f) After 0.6 s, 210 s, 630 s, 900 s, 1230 s excitation by laser (g) (i), (ii), (iii), (iv) mean signal of AuNRs, YO-PRO-1, transparent light and propidium iodide respectively. From Fig. (g), the apoptosis of the tumors can be observed.. . 61 .

(73) Fig. 3.9 Photothermolysis of the EMT-6 tumor cell triggered by PSS-AuNRs (N~2–15 clusters) under energy fluence 5.9 mJ/cm2. (a) Before excitation by laser, (b) After 0.6 s excitation by laser (e) (i), (ii), (iii), (iv) mean signal of AuNRs, YO-PRO-1, transparent light and propidium iodide respectively. From Fig. (e), after observation for 1180 s, YO-PRO-1 and propidium iodide do not penetrate the comparatively leaky membranes. It figures that that the tumor cell is still alive instead of destruction by photothermolysis.. Fig. 3.10 The situation of cancer cells to the power after photothermal therapy.. . 62 .

(74) 3.3 In vivo tumor photothermal therapy. 3.3.1 Power density test with the ears model In order to ensure that the photothermal effect as the dominant cell-killing factor, power density test is the first priority to exclude other possible influences towards tumor cell viability.. In. experiments, each ear of the mouse was exposed with continue-wave laser (808 nm) for 2 minutes with different power density, 1.2 W/cm2, 1 W/cm2, 0.8 W/cm2, and 0.6 W/cm2. results are shown in Fig. 3.11.. . 63 . The.

(75) Fig. 3.11 Power test of non-treated mice ears. (a) 1.2 W/cm2; (b) 1 W/cm2; (c) 0.8 W/cm2; (d) 0.6 W/cm2. From these results, the limited power density of 0.8 W/cm2 was observed obviously.. 3.3.2 Tumor growth curve To analyze the therapeutic effect of the PSS-AuNRs on photothermal ablation of solid tumors, the tumor (EMT-6 cells, 1*106 cells in 30 µl PBS) was induced by intratumoral injection on the ear of female mice (Balb/c). When the tumor size grew to approximately 3 mm in diameter, PSS-Au NRs (~10 µg) were directly injected into mice’s ears.. After 22~24 h, the mouse was. anesthetized with ether, and then the tumor region was irradiated. . 64 .

(76) with a laser for 2 minutes.. In order to ensure the photothermolysis. was the only factor that destroyed the solid tumors, setting relative control groups was necessary. There are three control groups: (i) tumors only, as shown in Fig. 3.12; (ii) tumors with exposure by 808 nm laser, 0.6 W/cm2, for 120 s, as shown in Fig. 3.13; (iii) tumors with PSS-AuNRs by intratumoral injection as shown in Fig. 3.14; and there is one experimental group with PSS-AuNRs and exposed under power density 0.6 W/cm2 for 120 s, as shown in Fig. 3.15. Fig. 3.12 Control group (tumors only). (a)~(f) are before induced tumors, and after induced for 5 days, 8 days, 11 days, 15 days, 20 days, respectively.. . 65 .

(77) Fig. 3.13 Control group (tumors exposed by laser). (a)~(f) are before induced tumors, and after induced for 5 days, 8 days, 11 days, 15 days, 20 days, respectively. Exposure with laser on the 12nd day, power density is 0.6 W/cm2, for 120 s. From this result, it can be observed that the laser was excluded as a factor, which destroys the solid tumors.. Fig. 3.14 Control group (tumors with PSS-AuNRs), Fig. (a)~(f) are after induced for 5 days, 8 days, 11 days, 15 days, 20 days, 24 days, respectively. 10 µg PSS-AuNRs were intratumorally injected into the solid tumor on the 11st day. From this result, it can be observed that the PSS-AuNRs was excluded as a cytotoxic factor.. . 66 .

(78) Fig. 3.15 Experimental group, the tumors were exposed with laser on the 12nd day, power density is 0.6 W/cm2, for 120 seconds. (a)~(g) are after induced for 5 days, 8 days, 11 days, 15 days, 20 days, 24 days, 28 days, respectively.. After calculation for the tumors size, the growth curve was presented in Fig. 3.16.. . 67 .

(79) Fig. 3.16 Growth Curve. The green-colored, blue-colored, black-colored and the red-colored symbol mean growth curve of tumors, tumors with laser, tumors with AuNRs and tumors with AuNRs under exposed by laser, respectively.. . 68 .

(80) 3.3.2 Histological study Histological sections of tumor tissues from each group confirmed the successful destruction of tumor cells by the photothermal effect by PSS-AuNRs. Fig. 3.16 (a)~(f) show the results of the each group.. . 69 .

(81) Fig. 3.17 Histological sections of tumor tissues (a) Normal cells (b) EMT-6 (c) EMT-6 under exposure with 0.6 w/cm2 laser for 2 min (d) EMT-6 with PSS-AuNRs injected (e) 1 day after photothermal treatment (f) 8 day after photothermal treatment. Fig. 3.17 shows that photothermal therapy can destroy the solid tumor structure and make severe destruction.. . 70 .

(82) Chapter 4 Conclusion All the experiments demonstrated the photothermal effect of PSS-AuNRs under NIR irradiation in destroying tumors cells in vitro and in vivo. The results of photothermal therapy in in vitro experiments indicated that the destruction of tumors under photothermal therapy were related to the energy fluence of radiation as well as the clusters of PSS-AuNRs that tumors took up. Under the same energy fluence, the more clusters tumors took up, the severer the damage; within the same clusters, the higher energy the power fluence was, the greater the damage. According to the above results, the energy fluence for photothermal therapy, controlled within the medical safety level (100 mJ/cm2), was dependent on the amount of AuNRs taken up per cell. When the solid tumors, where PSS-AuNRs were intratumorally injected, was irradiated by NIR laser, high thermal energy was generated. from. the. optical. excited. PSS-AuNRs,. which. subsequently destroyed tumor cells in a noninvasive manner. The ability of photothermal therapy to completely eradicate the tumor cells is evident in the results shown in the growth curve and . 71 .

(83) histological sections of the tumor, yet the great amount of heat will also damage normal cells, like cartilage. In future practices of photothermal therapy, better experimental conditions should be the first priorities, including variables such as power density, exposure time, concentration of AuNRs, and model size.. . 72 .

(84) . Reference [1] Cheng-Lung Chen1 and Yang-Yuan Chen. Ch.3, Nanomedicine and Cancer. 978-1-57808-727-3; January 2012; ca.370 pages incl. 42 color plates [2] Alexei P. Leonov, Jiwen Zheng, Jeffrey D. Clogston, Stephan T. Stern, Anil K. Patri, and Alexander Wei, Detoxification of Gold Nanorods by Treatment with Polystyrenesulfonate, ACS NANO, 2:12:2481-2488 (2008) [3] Jonathan A. Edgar, Andrew M. McDonagh, and Michael B. Cortie, Formation of Gold Nanorods by a Stochastic “Popcorn” Mechanism, ACS NANO, 6:2:1116-1125 (2012) [4] Adrian A. Ammann, Inductively coupled plasma mass spectrometry (ICP MS): a versatile tool, JMS 42: 419-427 (2007) [5] Mosmann T. Rapid colorimetric assay for cellular growth and survival: applicationto proliferation and cytotoxicity assays. J Immunol Methods 65:55–63. (1983). 73 .

(85) . [6] Cheng-Lung Chen, Ling-Ru Kuo, Ching-Lin Chang, Yeu-Kuang Hwua, Cheng-Kuang Huang, Shin-Yu Lee a, Kowa Chen Su-Jien Lin b, Jing-Duan Huang d, Yang-Yuan Chen a, In situ real-time investigation of cancer cell photothermolysis mediated by excited gold nanorod surface plasmons, Biomaterials 31:4014-4112(2010) [7] B. J. COVENTRY, P. J. MACARDLE, J. M. SKINNER AND J.BRADLEY, A technique for successful transplantation of tumors into ear-pouches of nude mice to maintain and study microenvironment, Surgical Oncology 3:127-129 (1994) [8] Chung-Yin Lin, Hsiao-Ching Tseng, Heng-Ruei Shiu Ming-Fang Wu, Cheng-Ying Chou, Win-Li Lin, Ultrasound sonication with microbubbles disrupts blood vessels and enhances tumor treatments of anticancer nanodrug International Journal of Nanomedicine 7:2143- 2152 (2012). 74 .

(86) . [9] Won Il Choi, Ja-Young Kim, Chul Kang, Clare C. Byeon, Young Ha Kim, and Giyoong Tae, Tumor Regression In Vivo by Photothermal Therapy Based on Gold-Nanorod-Loaded, Functional Nanocarriers, ACS NANO, 5:3:1995-2003 (2011) [10] Hye Kyung Moon, Sang Ho Lee, and Hee Cheul Choi, In Vivo Near-Infrared Mediated Tumor Destruction by Photothermal Effect of Carbon Nanotubes, ACS NANO, 3:11 3707-3713 (2009) [11] 李孟芳，張清風，黑鯛生殖系統的發育及調控, 國立海洋大學，水產養殖學系博士學位論文. 75 .

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以金奈米棒之光熱效應破壞癌細胞之活體 研究

以金奈米棒之光熱效應破壞癌細胞之活體研究