結合雙股去氧核醣核酸之磁性氧化鐵-金核殼奈米粒子的合成及特性分析用於可調節藥物釋放系統之研究

. . 國立交通大學 . . 材料科學與工程學系. 奈米科技碩士班. 碩士論文. . . . 結合雙股去氧核醣核酸之磁性氧化鐵-金核殼奈米粒子的合. 成及特性分析用於可調節藥物釋放系統之研究. . Synthesis and Characterization of Double Strand. DNA-Conjugated Magnetic Fe3O4 Core/Au Shell. Nanoparticles for a Tunable Drug Delivery System. 研究生：林庭伃 Ting–Yu Lin . 指導教授：柯富祥教授 Prof. Fu–Hsiang Ko. . . 中華民國一 ○ 一年八月. . . 結合雙股去氧核醣核酸之磁性氧化鐵-金核殼奈米粒子的合. 成及特性分析用於可調節藥物釋放系統之研究. Synthesis and Characterization of Double Strand DNA-Conjugated Magnetic Fe3O4 Core/Au Shell. Nanoparticles for a Tunable Drug Delivery System. 研究生：林庭伃 Student：Ting-Yu Lin. 指導教授：柯富祥教授 Advisor：Prof. Fu-Hsiang Ko. . 國立交通大學. 材料科學與工程學系. 奈米科技碩士班 . 碩士論文. . A Thesis. Submitted to Graduate Program for Nanotechnology. Department of Materials Science and Engineering. College of Engineering. National Chiao Tung University. in partial Fulfillment of the Requirements. for the Degree of. Master. in. Nanotechnology. August 2012. Hsinchu, Taiwan, Republic of China. 中華民國一○一年八月. . i . 結合雙股去氧核醣核酸之磁性氧化鐵-金核殼奈米粒子的合. 成及特性分析用於可調節藥物釋放系統之研究. 研究生:林庭伃指導教授:柯富祥教授. 國立交通大學材料科學與工程學系奈米科技碩士班. 摘要. 殼核雙金屬奈米粒子由兩種不同的材質所組成，與單一成分奈米粒子受限於. 單一性質相比，其更具新穎性及功能性。本篇研究以共沉澱法製備具良好分散性. 之奈米氧化鐵粒子，隨後以還原法在粒子表面沉積金殼層。並以穿透式電子顯微. 鏡以及選區電子繞射圖證實氧化鐵-金奈米粒子之核殼結構。此外，由紫外光-可. 見光吸收光譜以及超導量子干涉儀之量測，可證明氧化鐵-金奈米粒子具有金殼. 層之光學性質及氧化鐵核之超順磁特性。由於金殼層表面可與修飾有硫基的雙股. 去氧核醣核酸分子(DNA)作有效結合；同時利用一具有螢光特性的抗癌藥物「小. 紅莓」，可嵌入經設計的雙股去氧核醣核酸連續且重複的CG鹼基中，形成一創. 新且多功能的藥物載體。進一步地，將此磁性氧化鐵-金核殼奈米載體置於射頻. 磁場下，其磁性氧化鐵核因感應磁場而產生熱能，使得對熱敏感的雙股去氧核醣. 核酸解旋進而在短時間內釋放出藥物小紅莓。而藉由調整射頻磁場開啟的時間，. 便能調控藥物釋放的量。在本研究中，利用此一藥物釋放系統，在10分鐘內即可. 達到79%的釋放率。. 未來，此載體仍具有巨大發展潛力。金殼層表面可修飾專一性辨識分子的便. 利性，以及磁性氧化鐵核具有核磁共振顯影能力，加之無論是金或是氧化鐵，都. 已有文獻指出其高度生物相容性。假以時日，可使此磁性氧化鐵-金核殼奈米粒. . ii . 子的藥物釋放系統成為同時具有顯影能力、標靶治療，並且能調控藥物釋放的完. 整生醫奈米操作平台。. . iii . Synthesis and Characterization of Double Strand. DNA-Conjugated Magnetic Fe3O4 Core/Au Shell. Nanoparticles for a Tunable Drug Delivery System. Student: Ting–Yu Lin Advisor: Prof. Fu–Hsiang Ko. Institute of Nanotechnology. National Chiao Tung University. Abstract. Magnetic Fe3O4 core/Au shell nanoparticles exhibit both magnetic property of. Fe3O4 core and well-established surface chemistry, biological reactivity of Au shell.. In this study, magnetic Fe3O4 core/Au shell nanoparticles were synthesized by. reducing HAuCl4 on the surface of monodispersed Fe3O4 nanoparticles in the aqueous. solution after the facile synthesis of Fe3O4 nanoparticles by co-precipitation process. that are more popular for biological applications. Next, transmission electron. microscopy (TEM) images, selected-area electron diffraction (SAED) patterns, and. optical property by UV-Vis all clearly demonstrate the fact that core/shell. nanoparticles were successfully formed as the form of Fe3O4 nanoparticles covered by. Au shell; as well as superconducting quantum interference device (SQUID) results. show the great superparamagnetic property of Fe3O4 core/Au shell nanoparticles. In. addition, by functionalizing the surfaces of magnetic Fe3O4 core/Au shell. nanoparticles with CG rich double strand DNA through Au-S bonds, which are. thermo-sensitive molecules, the doxorubicin with fluorescence as anticancer drug. . iv . could be carried via intercalation. Further, when the innovative drug carriers were. heated under high frequency magnetic field, the double strand DNA dehybridized and. then caused the release of doxorubicin. Among suffered different periods of high. frequency magnetic field, the release of doxorubicin can be tuned. In this study, the. release rate can achieve up to 79% in less than 10 minutes based on our drug delivery. system. . As a result, this tunable drug delivery system with magnetic Fe3O4 core/Au shell. carriers has great promising of developing into multifunctional system including. imaging, targeting and tunable drug delivery owing to not only the potential in MR. imaging of magnetic core but also well-established surface with adaptive targeting. molecules of Au shell, even the proved biocompatibility whether it is Au or Fe3O4. nanoparticle. In the future, a complete bionano platform will be built up.. Key words: magnetic, core/shell, nanoparticles, drug delivery, doxorubicin. . v . Acknowledgment. 感謝柯富祥教授在學生的碩士生涯期間給予最充分的指導，並且提供豐富的. 資源與安全的實驗室環境，使我們能專心致志於研究以完成本篇論文。謝謝系主. 任陳三元教授與其實驗室的同學，提供高週波加熱器以及磁學上的指導。. 謝謝所有屬於柯富祥老師實驗室的學長姐、同學、學弟妹，不管是已經畢業. 或者是陪我一起度過碩班兩年生活，因為有你們當初的努力，才有今日的後人乘. 涼；因為有你們在實驗與報告上的指導與建議，才有我今日的進步；因為有你們. 平時的陪伴與關心，才使我的生活多彩多姿。. 最後感謝我最親愛的家人與朋友一路的陪伴與支持，在日常生活中關心我，. 在我遇到挫折時給予我鼓勵，使我可以無後顧之憂的完成碩班的學業，謝謝你們!. . vi . Contents. Abstract in Chinese ....................................................................................................... i . Abstract in English .................................................................................................... iii . Acknowledgment .......................................................................................................... v . Contents ....................................................................................................................... vi . List of Tables ............................................................................................................... ix . List of Figures ............................................................................................................... x. . Chapter 1: Introduction .............................................................................................. 1 . 1.1 General Introduction ......................................................................................... 1 . 1.2 Nanomaterials .................................................................................................... 1 . 1.3 Nanobiotechnology ............................................................................................. 5. . Chapter 2: Literatures Review & Motivation ........................................................... 8 . 2.1 Applications of Fe3O4 Superparamagnetic Nanoparticles ............................. 8 . 2.1.1 Magnetic Resonance Imaging .................................................................. 12 . 2.1.2 Drug Delivery ............................................................................................ 15 . 2.1.3 Hyperthermia ............................................................................................ 17 . 2.2 Core-Shell Nanoparticles................................................................................. 19 . 2.3 Doxorubicin ...................................................................................................... 21 . 2.4 Motivation ......................................................................................................... 23. . vii . Chapter 3: Experiments ............................................................................................ 25 . 3.1 General Introduction ....................................................................................... 25 . 3.2 Assaying the Specimen .................................................................................... 27 . 3.3 Experimental Methods .................................................................................... 30 . 3.3.1 Preparation of Fe3O4 Nanoparticles ........................................................ 31 . 3.3.2 Preparation of Fe3O4@Au Nanoparticles ............................................... 32 . 3.3.3 DNA Hybridization ................................................................................... 32 . 3.3.4 Fe3O4@AuNPs-Bound dsDNA ................................................................. 32 . 3.3.5 Doxorubicin-Intercalated Fe3O4@AuNPs-dsDNA ................................ 33 . 3.3.6 Drug Delivery of Fe3O4@AuNPs-dsDNA-Doxorbuicin at Various. Temperatures ..................................................................................................... 34 . 3.3.7 Drug Delivery of Fe3O4@AuNPs-dsDNA-Doxorbuicin under High. Frequency Magnetic Field ................................................................................. 34. . Chapter 4: Results and Discussion ........................................................................... 36 . 4.1 Synthesis and Characterization of Fe3O4 Nanoparticles and Fe3O4@Au. Nanoparticles .......................................................................................................... 36 . 4.1.1 Synthesis of Fe3O4 Nanoparticles and Fe3O4@Au Nanoparticles ........ 36 . 4.1.2 Zeta-potential of Fe3O4 Nanoparticles and Fe3O4@Au Nanoparticles 39 . 4.1.3 Size Distribution of Fe3O4 Nanoparticles and Fe3O4@Au Nanoparticles. .............................................................................................................................. 40 . 4.1.4 Characterization of Fe3O4 Nanoparticles and Fe3O4@Au Nanoparticles. . viii . .............................................................................................................................. 41 . 4.1.5 Magnetic Properties of Fe3O4 Nanoparticles and Fe3O4@Au. Nanoparticles ...................................................................................................... 45 . 4.2 Interaction between DNA Sequences and Doxorubicin ............................... 46 . 4.3 Fabrication of Drug Carrier—Fe3O4@AuNPs-dsDNA-Doxorubicin ......... 50 . 4.4 Drug Delivery Test ........................................................................................... 52 . 4.4.1 The Stability of Fe3O4@AuNPs-dsDNA-Doxorubicin at 37 oC ............. 53 . 4.4.2 Drug Release at Various Temperatures .................................................. 53 . 4.4.3 Drug Release under High Frequency Magnetic Field ........................... 55. . Chapter 5: Conclusions ............................................................................................. 58. . Reference .................................................................................................................... 59 . . ix . List of Tables. Table 2.1 ...................................................................................................................... 11. Summary comparison of Fe3O4 nanoparticles synthetic methods. [16]. Table 2.2 ...................................................................................................................... 17. Size-dependent heating rates of iron oxide nanoparticles for magnetic fluid. hyperthermia. Diameter measured with TEM, Chantrell method, polydispersity. determined from Chantrell method, concentrations during calorimetry measurement,. initial susceptibility and SLP measured at H0=24.5 kA/m for ferrofluid amples. [48]. Table 3.1 ...................................................................................................................... 27. The DNA sequences.. Table 4.1 ...................................................................................................................... 40. Zeta-potential of Fe3O4 nanoparticles in DI water, 0.01 MTMAOH, 0.1 M. THAOH, and Fe3O4@Au nanoparticles.. Table 4.2 ...................................................................................................................... 52 . Zeta-potential of Fe3O4@AuNPs, Fe3O4@AuNPs bound dsDNA, and. Fe3O4@AuNPs-dsDNA conjugated doxorubicin.. . x . List of Figures. Figure 1.1 ...................................................................................................................... 3. Sizes, shapes, and compositions of metal nanoparticles can be systematically. varied to produce materials with distinct light-scattering properties.[2]. Figure 1.2 ...................................................................................................................... 4. Surface spin canting effect of a nanoparticle upon magnetization (M magnetic. moment, H external magnetic field).[3]. Figure 1.3 ...................................................................................................................... 5. A gap currently exists in the engineering of small-scale devices. Whereas. conventional top-down processes hardly allow the production of structures smaller. than about 200 ± 100 nm, the limits of regular bottom-up processes are in the range of. about 2 ± 5 nm.[4]. Figure 1.4 ...................................................................................................................... 7. Integrated nanoparticle–biomolecule hybrid systems.[5]. Figure 2.1 ...................................................................................................................... 8. Magnetic nanoparticles: synthesis, protection, functionalization, and. application.[16]. Figure 2.2 ...................................................................................................................... 9. The reaction mechanism of Fe3O4 particle formation from an aqueous mixture of. ferrous and ferric chloride by addition of a base.[17]. . xi . Figure 2.3 .................................................................................................................... 10. The formation of Fe3O4 nanocrystals. The middle and right panels are TEM. images of the as-synthesized nanocrystals taken at different reaction times.[20]. Figure 2.4 .................................................................................................................... 11. The liduid-solid-solution (LSS) phase transfer synthetic strategy.[22]. Figure 2.5 .................................................................................................................... 13. MR contrast effect of magnetic nanoparticles (MNPs). Under an external field B0,. MNPs are magnetized with a magnetic moment of μ and generate an induced. magnetic field which perturbs the nuclear spin relaxation processes of the water. protons. This perturbation leads to MR contrast enhancement which appears as a. darkening of the corresponding section of the image.[26]. Figure 2.6 .................................................................................................................... 14. Important parameters of MNPs for MR contrast-enhancement effects.[26]. Figure 2.7 .................................................................................................................... 14. Tailored MNPs for molecular and cellular MR imaging. (a) Controlling the. magnetism of the nanoparticle core, (b) tailoring the surface ligands of the. nanoparticle shell and (c) the molecular targeting capability of. biomolecule-conjugated nanoparticles. (d) High performance utilizations of. nanoparticles for molecular and cellular MR imaging.[26]. Figure 2.8 .................................................................................................................... 16. Illustration of multifunctional imaging/therapeutic MNPs anatomy and potential. mechanisms of action at the cellular level. (A) A multifunctional MNP modified with. targeting ligands extended from MNP surface with polymeric extenders, imaging. . xii . reporters (optical, radio, magnetic), and potential therapeutic payloads (gene, radio,. chemo). (B) Four possible modes of action for various therapeutic agents; a) Specific. MNP binding to cell surface receptors (i.e. enzymes/proteins) facilitate their. internalization and/or inactivation, b) controlled intercellular release of. chemotherapeutics; c) release of gene therapeutic materials post endosomal escape. and subsequent targeting of nucleus; and d) intracellular decay of radioactive. materials.[30]. Figure 2.9 .................................................................................................................... 19. Illustration of the two components of the magnetic relaxation of a magnetic. fluid.[51]. Figure 2.10 .................................................................................................................. 20. Absorption (a) and photoluminescence (b) spectra of CdSe core and CdSe/ZnSe. core/shell nanocrystals before (dashed lines, toluene solutions) and after (solid lines,. aqueous solutions) functionalization with MUA. All colloidal solutions exhibit. identical optical densities at the first exciton absorption peak.[52]. Figure 2.11 .................................................................................................................. 21. (A) Mercaptoalkyl-oligonuleotide-modified Ag core/Au shell particles and. polynucleotide target. DNA spot test using: (B) 12.4 nm Ag/Au nanoparticle probes. and (C) 13 nm Au nanoparticle probes: (I) without target, (II) with target at room. temperature, (III) with target at 58.0oC, a temperature above the Tm (53.0 oC) of the. hybridized DNA.[53]. Figure 2.12 .................................................................................................................. 21. Thiol and carboxy modified γ-Fe2O3 beads are reacted with CdSe/ZnS QDs to. form the luminescent/magnetic nanocomposite particles.[54]. . xiii . Figure 2.13 .................................................................................................................. 22. Structure of doxorubicin.[55]. Figure 2.14 .................................................................................................................. 24. A multifunctional system including imaging, targeting, and tunable drug delivery. could be integrated into a Fe3O4 core/Au shell nanoparticle.. Figure 3.1 .................................................................................................................... 31. The experimental flowchart.. Figure 3.2 .................................................................................................................... 33. Schematic steps for the preparation of Fe3O4@AuNPs-bound dsDNA.. Figure 3.3 .................................................................................................................... 33. Schematic steps for the preparation of doxorubicin-intercalated. Fe3O4@AuNPs-dsDNA.. Figure 3.4 .................................................................................................................... 35. (a) Equipment of drug release induced by high frequency magnetic field. (b) The. sample was put in the center of the loop without any contact.. Figure 4.1 .................................................................................................................... 37. (a) Fe3O4 nanoparticles synthesized by co-precipitation showed a black color,. and (b) were concentrated by a magnet.. Figure 4.2 .................................................................................................................... 37. SEM images of Fe3O4 nanoparticles in different solvents: (a) DI water, (b) 0.01. M TMAOH, (c) 0.1 M TMAOH, (d) 0.1 M TMAOH with sonication.. . xiv . Figure 4.3 .................................................................................................................... 39. (a) Au shell processed Fe3O4 nanoparticles showed a pink color, and (b) were. concentrated by a magnet.. Figure 4.4 .................................................................................................................... 39. (a) SEM image of the Fe3O4 nanoparticles after coating Au shell. (b) UV-Vis. spectrum of Fe3O4 nanoparticles (dash) and Fe3O4 nanoparticles after coating Au shell. (solid).. Figure 4.5 .................................................................................................................... 41. Size distributions of (a) Fe3O4 nanoparticles, (b) Fe3O4@Au nanoparticles.. Figure 4.6 .................................................................................................................... 42. (a) TEM image of the Fe3O4 nanoparticles processed coating Au shell. (b) The. particles exhibited core/shell morphology.. Figure 4.7 .................................................................................................................... 43. EDS spectrum of the sample used to obtain Figure 4.6.. Figure 4.8 .................................................................................................................... 44. XRD spectra of (a) Fe3O4 nanoparticles, and (b) Fe3O4@Au nanoparticles.. Figure 4.9 .................................................................................................................... 45. SAED patterns of (a) Fe3O4 nanoparticles, and (b) Fe3O4@Au nanoparticles.. Figure 4.10 .................................................................................................................. 46. Magnetic hysteresis loops of Fe3O4 nanoparticles (square) and Fe3O4@Au. nanoparticles (circle).. . xv . Figure 4.11 .................................................................................................................. 47. Relationships between concentrations and fluorescence intensity of doxorubicin.. Figure 4.12 .................................................................................................................. 48. Fluorescence spectrum of a doxocrubicin solution (8.6 μM) with increasing. concentrations of ssDNA and dsDNA oligonucleotides.. Figure 4.13 .................................................................................................................. 49. Fluorescence spectrum of a doxocrubicin solution (8.6 μM) as a function of. temperature.. Figure 4.14 .................................................................................................................. 50. Fluorescence spectrum of a doxocrubicin solution (8.6 μM) with 125 nM dsDNA. as a function of temperature.. Figure 4.15 .................................................................................................................. 51. Photographic image of (a) Fe3O4@AuNPs, (b) Fe3O4@AuNPs after adding NaCl. solution, and (1)-(10) Fe3O4@AuNPs with decreasing concentrations of dsDNA after. adding NaCl solution.. Figure 4.16 .................................................................................................................. 53. The release profile of doxorubicin from doxorubicin-loaded. Fe3O4@AuNPs-dsDNA by diffusion at 37 oC.. Figure 4.17 .................................................................................................................. 54. Temperature-dependent release profile of doxorubicin from doxorubicin-loaded. Fe3O4@AuNPs-dsDNA.. . xvi . Figure 4.18 .................................................................................................................. 56. The fluorescence intensity of doxorubicin from doxorubicin-loaded. Fe3O4@AuNPs-dsDNA under high frequency magnetic field for different time. periods.. Figure 4.19 .................................................................................................................. 57. The release profile of doxorubicin from doxorubicin-loaded. Fe3O4@AuNPs-dsDNA under high frequency magnetic field for different time. periods.. . 1 . Chapter 1: Introduction. 1.1 General Introduction. “There’s plenty of room at the bottom,” an invitation to enter a new field of. physics, is the title of a classic speech given by the great physicist Richard Feynman. at the annual meeting of the American Physical Society at the California Institute of. Technology on December 29, 1959. Over 40 years ago, Feynman considered a new. physical world of ultra-small volumes and highlighted some difficulties that. researchers might confront when visiting it. His speech provided a vision for scientists. and engineers to establish a new field, which—with subsequent developments in. novel manufacturing skills and equipment—is now known as “nanotechnology.”. Nanotechnology has become one of the most important and interesting forefront. field in physics, chemistry, biology and engineering which the characteristic. dimensions are below ca. 1000 nm in recent years. It shows grand promise for. providing us in the near future with numerous breakthroughs that will change the. progress of technological advances in a wide range of applications. This kind of work. is often referred to as nanotechnology.. 1.2 Nanomaterials. An atom measures about 1 Ǻ, or 10-10 meters. The study of atoms and molecules. is the traditional field of chemistry as was researched in the late 19th and 20th. centuries. A nanometer (nm), or 10-9 meters, represent a collection of small number. atoms or molecules. Properties of bulk substances of micrometer sizes or larger have. been researched for years by solid state physicists and material scientists and are. currently well understood. Materials on the 1-100 nm scale were not studied by either. . 2 . group in the past. It was just recently shown that on this size scale the properties of a. material become dependent on its shape and size. Therefore, the nanometer scale. incorporates collections of molecules or atoms, whose properties are neither those of. the bulk nor those of the individual components. On this scale, numerous atoms are. still situated on the surface, or one layer removed from the surface, rather than the. interior. Different properties are seen on this scale due to the interface that is not seen. in the bulk or individual atom. Since the properties depend on the size of the structure,. instead of the nature of the material, continual and reliable change can be realized. using a single material.[1]. As the size reducing to the nano-regeime, a variety of properties different from. the bulk emerge, including optical, thermal, magnetic, and mechanical properties were. shown. The optical properties such as light absorption or microwave absorption are. significantly increased, and the absorption peak of Plasmon Resonance shifted which. creates the new optical character, such as infrared absorption and emission but a. sheltered role in ultraviolet, as well as the different size of metal particles with distinct. light-scattering properties (Figure 1.1).. . 3 . Figure 1.1 Sizes, shapes, and compositions of metal nanoparticles can be. systematically varied to produce materials with distinct light-scattering properties.[2]. Nanoparticles have small thermal resistance and excellent thermal conductivity. at low temperature, and can be used as a low-temperature thermal conductivity of. materials, because the amplitude of the nanomaterials surface atoms is twice as much. as the internal atoms. With the particle size decreases, the proportion of surface atoms. is gradually increased, and the melting point of nanomaterials will reduce,. distinguished the thermal properties. Moreover, when a magnetic material size. decreased, its magnetic susceptibility with decreasing temperature is gradually. reduced. The magnetically ordered state turns into a magnetic disordered state, and. the superconducting phase transfers to the normal phase; therefore some new. magnetic properties will generate (Figure 1.2). In addition, the strength, wear. resistance, aging resistance, pressure resistance, toughness, tight and waterproof. characteristic of nanocomposites are greatly increased and improved due to the. inadequate coordination surface atoms coupling with a very strong van der Walls fore.. Thus, some different mechanical properties are exhibited.. . 4 . Figure 1.2 Surface spin canting effect of a nanoparticle upon magnetization (M. magnetic moment, H external magnetic field).[3]. Besides, there are some physical effects appeared when the materials reduced to. nano size, making several properties different from the bulk materials such as the light. absorption, coercive force and melting point, called small size effect. The surface. atom of nanomaterials is increased, which makes high ratio of surface-to-volume, and. increases the surface energy, which was known as surface effect. Quantum size effect. occurs when the valence band and energy band gap of metal or semiconductor. nanomaterials widen which induce the insulation. Some nanoparticles having the. ability to run through the energy barrier is quantum tunneling effect. And some. nanomaterials show coulomb blockade effect, such as metal and semiconductor. showing that the charge and discharge process is not a continuous effect. In other. words, the current with the voltage increase is no longer straight up, but stepped up.. Along with synthetic advances for varying the composition, shape, and size of. nanostructured materials has come the most competitive candidate in various. applications.. . 5 . 1.3 Nanobiotechnology. Nanomaterials, such as semiconductor or metal nanorod and nanoparicles, show. similar dimensions to those of biomolecules, such as DNA or RNA or proteins. Both. materials science and biotechnology meet at the same length scale (Figure 1.3). On. one hand, biomolecular constituents have typical size dimensions in the range of. about 5 to 200 nm; on the other hand, commercial requirements to produce. increasingly miniaturized microelectronic devices strongly motivate the elaboration of. nanoscale systems. Today’s nanotechnology research puts a grand importance on the. development of bottom-up strategies, which interest the self-assembly of (macro). molecular and colloidal building blocks to create larger, functional devices.. Figure 1.3 A gap currently exists in the engineering of small-scale devices. Whereas. conventional top-down processes hardly allow the production of structures smaller. than about 200±100 nm, the limits of regular bottom-up processes are in the range of. about 2±5 nm.[4]. The combination of nanomaterials and biotechnology has led to the development. of the hybrid nanomaterials that combine the highly selective catalytic and. . 6 . recognition properties of biomaterials, such as DNA and protein/enzymes, with. unique photonic, electronic, and catalytic features of nanoparticles. Because of many. fundamental features, biomaterials are major future building blocks for nanomaterials. architectures. Biomaterials play the roles of specific and strong complementary. recognition interactions, for example, nucleic acid–DNA antigen–antibody, and. hormone–receptor interactions. The functionalization of nanomaterials with. biomolecules could lead to biomolecule-nanoparticle recognition interactions and thus. to self-assembly. In addition, numerous biomolecules contain several binding sites.. First, for example, antibodies show two Fab (antigen-binding fragment) sites, whereas. streptavidin or concanavalin A each exhibit four binding domains. This allows the. multidirectional growth of nanomaterial structures. Second, proteins may be. genetically engineered and modified with specific anchoring groups. This assists their. aligned binding to nanomaterials or the site-specific linkage of the biomaterial to. surfaces. As the result, the directional growth of nanomaterial structures may be. instructed. Furthermore, other biomolecules, such as double-stranded DNA, may be. synthetically produced in complex rigidified structures that play the role of templates. for the assembly of nanomaterials by intercalation, electrostatic binding to phosphate. groups, or association to functionalities tethered to the DNA. Next, enzymes are. catalytic instruments for the manipulation of biomaterials. For example, the. endonuclease scission processes of nucleic acids or the ligation provide effective. instruments for controlling the structure and shape of biomolecule-nanomaterial. hybrid systems. In this context, it is important to notice that Mother Nature has. processed unique biocatalytic replication developments. The use of biocatalysts for. the replication of biomolecule-nanomaterial conjugates may provide a productive. system for the production of nanostructures of predesigned compositions and. shapes.[5-6] In this regard, the conjugation of nanomaterials (e.g. nanoparticles,. . 7 . nanorods, nanowires) with biomolecules (e.g. DNA, RNA, proteins) is a desirable. area of research within nanobiotechnology.. Biomolecule-functionalized nanomaterials could be exploited for abundant. applications in biomolecular biosensors,[7-8] immunoassays,[9] medicine,[10] and. electronics,[11-12] namely in drug delivery,[13] photodynamic anticancer therapy,. electronic DNA sequencing, targeted delivery of radioisotopes, gene therapy, and. nanotechnology of gene-delivery systems in Figure 1.4. Novel fascinating regions of. technologies are practical with the use of bionanomaterials. A combination of the. unique properties of biomaterials and nanomaterials provides a unique chance for. chemists, physicists, biologists, and material scientists to shape the new region of. nanobiotechnology.[14] Based on recent progress in the field, invigorating new science. and novel system can be anticipated from the interdisciplinary effort. Future progress. will need continued innovation by nanotechnology in close cooperation with experts. in biological and medical fields.. Figure 1.4 Integrated nanoparticle–biomolecule hybrid systems.[5]. . 8 . Chapter 2: Literatures Review &. Motivation. 2.1 Applications of Fe3O4 Superparamagnetic Nanoparticles. Superparamagnetic Fe3O4 nanoparticle with proper surface chemistry have been. extensively used for several in vivo applications, such as drug delivery, cell separation,. immunoassay, tissue repair, detoxification of biological fluids, and magnetic. resonance (MR) imaging contrast enhancement (Figure 2.1).[15] Because of its unique. characters including degradable innocuously, manipulated by an external magnetic. field, and heated by high frequency magnetic fields, which is called hyperthermia. It. is notable that all of these bioengineering and biomedical applications require that the. nanoparticles have a narrow particle size distribution, a size smaller than 100 nm, and. high magnetization values, so that the nanoparticles have uniform chemical and. physical properties. . Figure 2.1 Magnetic nanoparticles: synthesis, protection, functionalization, and. application.[16]. . 9 . There are several popular methods to synthesize monodispersed, highly stable,. and shape-controlled magnetic nanoparticles, including co-precipitation, thermal. decomposition, microemulsion, and hydrothermal synthesis. There follows each. method respectively.. Co-precipitation is a convenient and facile way to synthesize Fe3O4 nanoparticles,. which prepared under atmosphere at room temperature. The chemical reaction of. Fe3O4 co-precipitation is given in Figure 2.2. By modulating type of salt used,. reaction temperature, Fe2+/Fe3+ ratio, ionic strength of the media, and pH value,. different shape, size, and composition of magnetic nanoparticles are altered.[17]. However, nanoparticles synthesized by this method tend to be rather polydispersed.. Figure 2.2 The reaction mechanism of Fe3O4 particle formation from an aqueous. mixture of ferrous and ferric chloride by addition of a base.[17]. Fe3O4 nanoparticles by thermal decomposition synthesized are usually. synthesized with a narrow size through the organometallic compounds in high-boiling. organic solvents with stabilizing surfactants (Figure 2.3).[18-19] The reaction. temperature, aging period, and the ration of the starting reagents are the determining. . 10 . parameters for the control of the morphology and the size of magnetic nanoparticles.. The reactivity was tuned by altering the concentration and the chain length of fatty. acids. However, it is unclear whether the magnetic nanoparticles can be dispersed in. water, so the water-soluble magnetic nanoparticles are more popular for biological. applications.. Figure 2.3 The formation of Fe3O4 nanocrystals. The middle and right panels are. TEM images of the as-synthesized nanocrystals taken at different reaction times.[20]. In addition to the above-mentioned two methods, microemulsion is a. thermodynamically stable isotropic dispersion of two immiscible liquids, where the. microdomain of either or both liquids has been stabilized by an interfacial film of. surfactant molecules. Nevertheless, the shape and size of the Fe3O4 nanoparticles. usually vary over a relatively wide range.[21]. Hydrothermal synthesis is a liquid-solid-solution reaction which is more. complicated and expensive. During reaction process, a variety of different. nanocrystals have been synthesized. This system consists of solid (metal linoleate), a. liquid phase (ethanol-linoleic acid), and a solution (water-ethanol) at different. reaction temperatures under hydrothermal conditions (Figure 2.4).[22] In this way,. Fe3O4 nanopaeticles were obtained with adjustable sizes in the range of 200-800 nm.. In the following, Table 2.1 shows the comparison of the advantages and. disadvantages of the four above-mentioned methods.. . 11 . Figure 2.4 The liduid-solid-solution (LSS) phase transfer synthetic strategy.[22]. Table 2.1 Summary comparison of Fe3O4 nanoparticles synthetic methods. [16]. The stability of Fe3O4 nanoparticles is important for the preparation and storage.. With appropriate surface coating, Fe3O4 nanoparticles can be dispersed into suitable. solvents, forming ferrofluids. Nanosized particles have chemical and physical. properties that are characteristic of neither the bulk nor the atom.[23] The large surface. area and quantum size effects of Fe3O4 nanoparticles dramatically alter some of the. magnetic properties and show superparamagnetic phenomena, because each particle. can be seen as a single magnetic domain.[24] Based on their unique nature, Fe3O4. superparamagnetic nanoparticles have been widely used in biological applications.. . 12 . 2.1.1 Magnetic Resonance Imaging. Magnetic resonance (MR) imaging is one of the greatest non-invasive imaging. modalities used in clinical medicine today, which based on the property that hydrogen. protons will align and process around an applied magnetic field, B0. Upon applying a. transverse radiofrequency (rf) pulse, the protons are perturbed from B0. The following. process through which the protons return to their original state is called the relaxation. phenomenon. There are two independent process, longitudinal relaxation (T1) and. transverse relaxation (T2), which can be monitored to create an MR imaging. The. local variation of relaxation is corresponding to imaging contrast, which arises from. proton density as well as the physical and chemical properties of the tissues.[25]. Superparamagnetic nanoparticles play a magnificent role as MR imaging. contrast agents, to better distinguish healthy and pathological tissues. Under an. applied B0, superparamagnetic nanoparticles induce a magnetic dipole moment μ.. When water molecules diffuse into the periphery of the induced μ, the magnetic. relaxation processes of the water protons are perturbed and T2 is shorted, which result. in the darkening of the corresponding area in T2-weighted MR imaging (Figure 2.5).. The degree of the T2 contrast effect is illustrated by the spin-spin relaxivity R2 (1/T2).. The higher values of R2 result in a better contrast effect.. . 13 . Figure 2.5 MR contrast effect of magnetic nanoparticles (MNPs). Under an external. field B0, MNPs are magnetized with a magnetic moment of μ and generate an induced. magnetic field which perturbs the nuclear spin relaxation processes of the water. protons. This perturbation leads to MR contrast enhancement which appears as a. darkening of the corresponding section of the image.[26]. The control of nanoparticle magnetism should be led to a maximum R2 value.. Therefore, by modulating the nanoparticle parameters, synthetic magnetic. nanoparticles can be designed and constructed to raise the MR contrast-enhancement. effects (Figure 2.6). Moreover, controlling the magnetic core, modifying the ligand. shell to reach high colloidal stability and biocompatibility, and conjugating the. biomolecules to get the molecular targeting capability, can make the nanoparticles for. MR imaging of cancer, cellular trafficking, angiogensis, and therapy (Figure 2.7).[26]. . 14 . Figure 2.6 Important parameters of MNPs for MR contrast-enhancement effects.[26]. Figure 2.7 Tailored MNPs for molecular and cellular MR imaging. (a) Controlling the. magnetism of the nanoparticle core, (b) tailoring the surface ligands of the. nanoparticle shell and (c) the molecular targeting capability of. biomolecule-conjugated nanoparticles. (d) High performance utilizations of. nanoparticles for molecular and cellular MR imaging.[26]. . 15 . 2.1.2 Drug Delivery. Another possible and most favorable application of Fe3O4 nanoparticles is in. drug delivery as vehicle of therapeutic drug for targeting delivery. When packing a. therapeutic drug and with a proper design, Fe3O4 nanoparticles can act as efficient. drug delivery systems, which offer a variety of drug loaded, control the drug release,. and track the delivery by using Fe3O4 nanoparticles imaging modality. There are. several factors, such as the size, charge and surface chemistry of Fe3O4 nanoparticles,. particularly and strongly affect both the blood circulation and biocompatibility of. magnetic nanoparticles within the body.[27]. Developments of Fe3O4 nanoparticles for drug-carrying with diagnostic imaging. not only require precision physical and chemical design, but also consider the pay of. drug loading, transport, and release.[28-29] First, the coating type and loading way of. Fe3O4 nanoparticles determine the abilities of carrying and protecting a significant. drug payload. Second, Fe3O4 nanoparticles carefully loading with multiple drugs to. accommodate the diverse therapeutics can overcome the cellular resistance, and. increase the overall cell kill efficiencies. Third, the release mechanism and rate of the. drug delivery system should be modulated for optimal therapeutic capability.[30] Since. the large surface to volume ratio, Fe3O4 nanoparticles tend to agglomerate and absorb. plasma proteins. To avoid these phenomena, coating with amphiphilic polymeric. surfactants (poloxamers, poloxamines, and PEG) can stabilize nanoparticles and. prolong the blood circulation time.[31] Moreover, modifying the targeting ligands. increases the specific accumulation of nanoparticles within diseased tissue. By. loading with different types of drugs, such as chemotherapeutic,[32-35] peptides and. proteins,[36-38] DNA and siRNA,[39-44] Fe3O4 nanoparticles can apply to a wide range. of diseases. For instance, Fe3O4 nanoparticles loading with chemotherapeutic agents,. such as doxorubicin, etoposide, and methotrexate, have been used in breast and. . 16 . prostate tumor therapy. HerceptinTM, a protein, has been conjugated to magnetic. nanoparticles as a mAb targeting molecule, which exhibits a therapeutic effect by. causing cells to steady in G1 phase of the cell cycle and thereby reduces cell. proliferation. Fe3O4 nanoparticles conjugated with antisense oligodioxynucleotides for. gene therapy significantly increase the half-time in vivo. Figure 2.8 shows the. blueprint of multifunctional imaging/therapeutic magnetic nanoparticles and the local. activity of several classes of drugs for cancer therapy.. Figure 2.8 Illustration of multifunctional imaging/therapeutic MNPs anatomy and. potential mechanisms of action at the cellular level. (A) A multifunctional MNP. modified with targeting ligands extended from MNP surface with polymeric extenders,. imaging reporters (optical, radio, magnetic), and potential therapeutic payloads (gene,. radio, chemo). (B) Four possible modes of action for various therapeutic agents; a). Specific MNP binding to cell surface receptors (i.e. enzymes/proteins) facilitate their. internalization and/or inactivation, b) controlled intercellular release of. chemotherapeutics; c) release of gene therapeutic materials post endosomal escape. and subsequent targeting of nucleus; and d) intracellular decay of radioactive. materials.[30]. . 17 . 2.1.3 Hyperthermia. Although Fe3O4 nanoparticles don’t carry any drugs, they still have therapeutic. effects by themselves. Under high frequency magnetic field, Fe3O4 nanoparticles can. generate heat energy due to the spin vibration by themselves, which is called. hyperthermia. Since tumors are more sensitive to a temperature increase than healthy. ones, the hyperthermia of Fe3O4 nanoparticles can be utilized to increase the. temperature of tumor cells and thereby destroy the pathological cells.[45-46] The. application of hyperthermia was first envisaged by Jordan et al. in 1993.[47] The. amount of heat generated determines by the properties of magnetic nanoparticles and. magnetic field parameters. For example, Marcela et al. have demonstrated that the. heating rate of Fe3O4 nanoparticles are dependent on particle size at the same. frequency magnetic field (Table 2.2).[48]. The main parameter determining the temperature increasing of the ferrofluid. sample is the specific loss power (SLP), which is defined as the thermal power. dissipation divided by the mass of magnetic nanoparticles and can be written as. SLP d d. where C is the specific heat capacity of the sample, Vs is the sample volume, m is the. mass of magnetic nanoparticles in the sample.. Table 2.2 Size-dependent heating rates of iron oxide nanoparticles for magnetic fluid. hyperthermia. Diameter measured with TEM, Chantrell method, polydispersity. determined from Chantrell method, concentrations during calorimetry measurement,. initial susceptibility and SLP measured at H0=24.5 kA/m for ferrofluid amples. [48]. . 18 . The heating effects of magnetic nanoparticles under a high frequency field are. due to several types of loss processes, like hysteresis losses, Néel and Brown. relaxation (Figure 2.9), the relative contributions of which depend strongly on the. particle size. Magnetic nanoparticles with a diameter less than 30 nm are. single-domain particles. Accordingly, their heat effects are governed by the. combination of the rotational internal (Néel) and external (Brown) diffusion of. particle magnetic moment.[49] Néel relaxation is due to thermal rotation of particle’s. magnetic moment within the crystal, which takes place when the anisotropy energy. barrier (Ea=KV, where K the is anisotropy of the magnetic nanoparticles, V is the. magnetic nanoparticls volume) is overcome. The characteristic time τN for Néel. relaxation is expressed as τN=τ0 exp (Ea/KV), where τ0 is of the order of 10 -9 s. Brown. relaxation refers to thermal orientational fluctuations of the particle itself in the carrier. fluid, the magnetic moment being locked onto the crystal anisotropy axis. The. characteristic time τB for Brown relaxation is expressed as τB=3ηVH/kT, where η is. the viscosity of the carrier fluid, VH is the hydrodynamic volume of the particle, k is. the Boltzmann constant, T is the temperature. When the two relaxation mechanisms. occur in parallel, the effective relaxation time τ is given by the relationship:. 1/τ=1/τN+1/τB. Note that the shorter time determines the dominant mechanism of. relaxation.[50]. . 19 . Figure 2.9 Illustration of the two components of the magnetic relaxation of a. magnetic fluid.[51]. 2.2 Core-Shell Nanoparticles. A core-shell nanoparticle is broadly defined as core and shell of different. materials in close interaction, including inorganic, organic, biological core-shell. combination. There are numerous advantages make core-shell nanoparticle playing an. important role in material chemistry, including (1) size monodispersity, (2) core and. shell processibility, (3) stability, (4) solubility, (5) tunability, (6) self-assembling. capability, (7) reactivities involving in chemical, biological, magnetic, optical, electric,. and catalytic phenomena. Consequently, core-shell nanoparticles have a variety of. application range from magnetic, quantum dots, microelectronic, and photoactive. devices.. Following are some examples of various types of core-shell nanoparticles. The. monodispersed CdSe nanocrystals coating with a ZnSe shell show high room. temperature photoluminescence efficiencies (60-85%) in organic solvents as well as. in water after modification with mercaptoundecanoic acid (MUA) (Figure 2.10).[52]. . 20 . Figure 2.10 Absorption (a) and photoluminescence (b) spectra of CdSe core and. CdSe/ZnSe core/shell nanocrystals before (dashed lines, toluene solutions) and after. (solid lines, aqueous solutions) functionalization with MUA. All colloidal solutions. exhibit identical optical densities at the first exciton absorption peak.[52]. An Ag core with Au shell nanoparticle, which exhibits a Surface Plasmon band. between 390 nm and 420 nm with larger extinction coefficient of sliver but the. stability and surface chemistry of gold, has been used to access a colorimetric. detection system distinct from a pure gold system (Figure 2.11).[53] The luminescent. and magnetic nanoparticles, which were easily separated from solution by magnetic. decantation using a permanent magnet, have been synthesized by coating CdSe/ZnS. shell on γ-Fe2O3 superparamagnetic core (Figure 2.12). [54] Due to the various. properties of each materials, someone can synthesize a novel material for their own. use by combining different materials into a core-shell nanoparticle.. . 21 . Figure 2.11 (A) Mercaptoalkyl-oligonuleotide-modified Ag core/Au shell particles. and polynucleotide target. DNA spot test using: (B) 12.4 nm Ag/Au nanoparticle. probes and (C) 13 nm Au nanoparticle probes: (I) without target, (II) with target at. room temperature, (III) with target at 58.0oC, a temperature above the Tm (53.0 oC) of. the hybridized DNA.[53]. Figure 2.12 Thiol and carboxy modified γ-Fe2O3 beads are reacted with CdSe/ZnS. QDs to form the luminescent/magnetic nanocomposite particles.[54]. 2.3 Doxorubicin. Cancer, one of the most terrible diseases, is caused by DNA mutation,. rearrangement, and deletion, which introduce oncogenes to promote cell irregularly. growth and reproduction. Thus, developing of anticancer drugs are usually focusing. on the interaction with DNA molecules, such as DNA alkylation agent, intercalating. agent, cleaving agent, and binding agent. After interacting with DNA molecules, the. anticancer drugs affect the DNA replication, transcription, and translation.. . 22 . Doxorubicin (Figure 2.13) is one of anthracyclin antibiotics, which are the most. powerful weapons in the chemical arsenal used for cancer chemotherapy. The binding. mechanisms contain (1) the planar anthraquinone ring system intercalating into DNA,. (2) the puckered anchor D ring giving additional contacts to DNA via direct and. indirect hydrogen-bonding interactions, and (3) the daunosamine sugar playing a role. in a minor groove binder and recognition of the DNA base surface within the helical. groove.[56] When doxorubicin binding with DNA, it will stabilize the topoisomerase. Ⅱ complex, keeping the DNA double helix from separating and thereby stopping the. process of replication.. Doxorubicin has been commonly used in the treatments of a wide range of. cancers, including breast cancer, lung cancer, hematological malignancies, and soft. tissue sarcomas. The normal dosage is 20 mg/m2 every week or 60-75 mg/m2 every 3. weeks.[57] However, high accumulative doses of doxorubicin increase the probability. of cardiotoxicity (e.g. 550 mg/m2 of accumulative doxorubicin has 7% probability of. cardiotoxicity).[58] Therefore, exploring a escort of delivery molecule to reduce the. side effects is necessary.. Figure 2.13 Structure of doxorubicin.[55]. . 23 . 2.4 Motivation. Cancer has threatened the lives of humankind for many years. Despite the fact. that the cure for cancer had taken effect in recent years, repeated diagnosis (e.g.. magnetic resonance imaging, ultrasound, computed tomography, and positron. emission tomography) and treatments (e.g. surgery, chemotherapy, and radiation. therapy) have been bringing infinite sufferings to the patients instead of pains caused. by cancer itself. Therefore, developing a multifunctional pharmaceutical that can. simultaneously perform various functions, including targeting, imaging, and therapy,. is necessary.. As we all know, nanomaterials, such as quantum dots, superparamagnetic iron. oxide nanoparticles, and gold nanoparticles, have been investigated for potential. multifunctional purposes as imaging agents capable of visualization by MR imaging. or optical imaging techniques, therapeutic agents, and delivery vehicles. In the various. nanomaterials, superparamagnetic nanoparticles become more and more popular. owing to their potential in MR imaging which plays an important role in non-invasive. imaging. In addition, the magnetic property of magnetic nanoparticles make. themselves can be heated by high frequency magnetic fields and manipulated by an. external magnetic field. Thus, magnetic nanoparticles have been extensively used in. biomedical applications. Fe3O4 nanoparticle is one of the magnetic nanoparticles. which can be simply synthesized under atmosphere at room temperature by. co-precipitation. However, particles prepared by co-precipitation tend to be. polydispersed. In order to improve this phenomenon, introducing a gold shell around. them makes they more stable and highly monodispersed. In addition, gold surface is. in favor of functionalization with thiolated biomolecules.. By modifying double strand DNA, which are thermo-sensitive molecules and. can be denatured of the double helix to form two single strands, the anticancer drug,. . 24 . doxorubicin, can be carried by intercalation. When high frequency magnetic field. heated the superparamagnetic nanoparticles, the modified double strand DNA would. unwind and release the anticancer drugs. By adjusting duration time under high. frequency magnetic field, the drug delivery can be tuned. Therefore, a multifunctional. system including imaging, targeting, and tunable drug delivery could be integrated. into a Fe3O4 core/Au shell nanoparticle (Figure 2.14).. Figure 2.14 A multifunctional system including imaging, targeting, and tunable drug. delivery could be integrated into a Fe3O4 core/Au shell nanoparticle.. . 25 . Chapter 3: Experiments. 3.1 General Introduction. All the experiments were preceded in National Chiao Tung University (NCTU).. All the equipments were also conducted in our laboratories in NCTU. The reagents. were purchased commercially and used by following with the directions unless. specially mentioned.. All the reagents were listed alphabetically in the form of “Name {abbreviation;. chemical formula; purity; manufacturer}”. Some information will be omitted if not. available or not necessary. The following text will use the abbreviation of the reagent.. Deionized and distilled water {DI water, ddH2O}. The water we used was purified with filters, reverse osmosis, and deionized. system until the resistance was more than 18 MΩ‧cm-1. DI water was used to clean,. wash, and be a solvent.. Iron(Ⅱ) chloride tetrahydrate {FeCl2·4H2O, 98%; Alfa Aesar}. The chemical provided Fe2+ ion for the co-precipitation of Fe3O4 nanopaticles. synthesis.. Iron(Ⅲ) chloride hexahydrate {FeCl3·6H2O, 98%; Alfa Aesar}. The chemical provided Fe3+ ion for the co-precipitation of Fe3O4 nanopaticles. synthesis.. . 26 . Hydrogen chloride {HCl, ≥99% purity; Sigma}. It was used to dissolve iron(Ⅱ) chloride tetrahydrate and iron(Ⅲ) chloride. hexahydrate for Fe3O4 nanopaticles synthesis.. Sodium hydroxide {NaOH, 97%; SHOWA}. This pellets was dissolved in DI water to yield 3 M sodium hydroxide solution,. which provided the alkaline environment to prepared Fe3O4 nanopaticles by. co-precipitation of Fe(Ⅱ) and Fe(Ⅲ) chlorides in an alkaline solution.. Tetramethylammonium hydroxide {(CH3)4NOH, 25% w/w in aqueous solution;. Fluka }. This alkaline solution was diluted with DI water for various concentrations to. improve the dispersion of Fe3O4 nanoparticles.. Gold(Ⅲ) chloride trihydrate {HAuCl4·3H2O, ≥49%; Sigma}. The chemical was a provider of Au3+ ion for the mechanism of Au nanoshell. synthesis.. Trisodium citrate dihydrate {C6H5Na3O7·2H2O, 98%; SHOWA}. We used a previously reported chemical reduction method to prepare Au. nanoshell in aqueous solution. This chemical was a reductant of the reaction. Prior to. use, the powder needed to dissolve in the DI water.. Hydroxylamine hydrochloride {NH2OH·HCl, 99%; Alfa Aesar}. The powder was dissolved in the DI water, and was used in the reaction of. coating Au nanoshell onto the Fe3O4 nanopaticles.. . 27 . Drug delivery oligonucleotides. A short nucleic acid polymer, typically has fifty or fewer bases. In this study,. oligonucleotides were obtained from MDBio, Inc. (Taiwan) and hybridized to form. double strand DNA for drug carrying. The DNA sequences were showed in Table 3.1.. Name Sequence . T3 5'-CGA CGA CGA CGA CGA CGA TTT-3'. A15 SH-5'-AAA AAA AAA AAA AAA TCG TCG TCG TCG TCG TCG-3'. A15T3 5'-CGA CGA CGA CGA CGA CGA TTT-3' . 3'-GCT GCT GCT GCT GCT GCT AAA AAA AAA AAA AAA-5'-SH. Table 3.1 The DNA sequences.. Doxorubicin hydrochloride {C27H29NO11·HCl, ≥98%; Sigma}. This molecule played roles as both anticancer drug and fluorescence agent,. which had an exciting wavelength at 470 nm and an emission wavelength at 590 nm.. 3.2 Assaying the Specimen. (A) SEM. SEM is a very useful tool for observing surface morphology of specimen. SEM. has secondary electrons or backscattered electrons detectors passing the signal to. computer and forming image. In this study, the morphology and microstructure of the. Fe3O4 nanoparticles and Fe3O4 core/Au shell nanoparticles were all characterized by a. field-emission SEM (FE-SEM) (JEOL-6700) operating at 10 kV accelerating voltage.. . 28 . (B) XRD. X-ray powder diffraction (XRD) is a rapid analytical technique primarily used. for the study of crystal structures and atomic spacing. X-ray diffraction is based on. constructive interference of monochromatic X-rays and a crystalline sample. It can. show the phase identification of a crystalline material and can provide information on. unit cell dimensions. The analyzed material is finely ground, homogenized, and. average bulk composition is determined. The interaction of the incident rays with the. sample produces constructive interference (and a diffracted ray) when conditions. satisfy Bragg’s Law (nλ=2d sin θ). These diffracted X-rays are then detected,. processed and counted. By scanning the sample through a range of 2θ angles, all. possible diffraction directions of the lattice should be attained due to the random. orientation of the powdered material and then to converse the diffraction peaks to. d-spacings allows identification of the mineral because each mineral has a set of. unique d-spacings. In this study, the microstructure of the Fe3O4 nanoparticles and. Fe3O4 core/Au shell nanoparticles were all characterized by an aid of x-ray diffraction. (X'Pert PRO MRD system).[59]. (C) TEM. Transmission electron microscopy (TEM) is a microscopy technique. By. shooting an electronic beam to transmit an ultra thin specimen, it can observe the. internal morphology and crystal atomic structure of specimen. In this study, TEM. (JEOL, JEM-2010) was used to examine the internal morphology and electron. diffraction of the Fe3O4 nanoparticles and Fe3O4 core/Au shell nanoparticles.. . 29 . (D) EDS. Energy dispersive X-ray spectroscopy (EDS) is an analytical technique used. for the elemental analysis or chemical characterization of a sample. It relies on the. investigation of a sample through interactions between electromagnetic radiation and. matter, analyzing x-rays emitted by the matter in response to being hit with charged. particles. Its characterization capabilities are due in large part to the fundamental. principle that each element has a unique atomic structure allowing x-rays that are. characteristic of an element's atomic structure to be identified uniquely from each. other.[60] In our experiment, EDX (Oxford-Link ISIS 300 energy-dispersive X-ray). give our analytical of elements for the specimen under TEM monitoring.. (E) SQUID. Superconducting quantum interference device (SQUID, MPMS-XL) is an ultra. sensitive magnetometer used to measure extremely subtle magnetic field of Fe3O4. nanoparticles and Fe3O4 core/Au shell nanoparticles, which based on superconducting. loops containing Josephson junctions.. (F) DelsaTM Nano Submicron Particle Size and Zeta Potential. Zeta potential is a scientific term for electronkinetic. The value of zeta potential,. which can be related to the stability, is the potential difference between the dispersion. medium and the stationary layer of fluid attached to the dispersed particle. The zeta. potential of Fe3O4 nanoparticles and Fe3O4 core/Au shell nanoparticles is measured by. DelsaTM Nano Submicron Particle Size and Zeta Potential (BECKMAN COULTER,. PN A54412AA) in this study.. . 30 . (G) UV-Vis Spectroscopy. UV-Vis spectroscopy uses light in the range of near UV, visible and near. infrared. The absorption in the light range is due to the optical properties of the. chemicals involved. We determine whether the Fe3O4 nanoparticles covered by Au. shell or not in the visible range of 400-800 nm by the UV-Vis spectroscopy. (HITACHI, U-3310).. (H) Fluorescence Spectroscopy. Fluorescence spectroscopy (HITACHI, F-7000) is used to measure the. doxorubicin release from Fe3O4 core/gold shell nanoparticle carrier by shooting a 470. nm light to excite the electrons in doxorubicin molecule and measuring the intensity. of 590 nm light emitting from the exciting electrons to determine the concentration of. doxorubicin.. (G) HFMF. High frequency magnetic field is a machine which can provide oscillating. magnetic field to heat the magnetic materials. In this study, high frequency magnetic. field (GE-15) with a frequency of 50 kHz and a magnetic field strength (H) of 8 kA/m. was used to heat Fe3O4 nanoparticles and Fe3O4 core/Au shell nanoparticles due to. Néel and Brown relaxation of superparamagnetic nanoparticles in oscillating. magnetic field.. 3.3 Experimental Methods. The synthetic flow includes synthesis of Fe3O4 nanoparticles, coating Au shell. onto Fe3O4 nanoparticles, DNA hybridization, modifying with double strand DNA,. intercalating doxorubicin, and drug release test (Figure 3.1).. . 31 . Figure 3.1 The experimental flowchart.. 3.3.1 Preparation of Fe3O4 Nanoparticles. The Fe3O4 nanoparticles were prepared in aqueous solution using a previously. reported co-precipitation method.[17] Firstly, 0.4 g of FeCl2·4H2O, 1.04 g of. FeCl3·6H2O and 170 μl 12 M HCl were dissolved in 5 mL of DI water for 30 min. with stirring. Secondly, the solution was added dropwise into 25 mL of 3 M NaOH. solution with vigorous stirring, and then a black precipitate formed immediately.. Finally, the precipitate was isolated by a mighty magnet, and washed twice with DI. water, and then resuspended in 0.1 M (CH3)4NOH solution. The black, 6.5 mg/mL. magnetic nanoparticle solution was stored in air under benchtop conditions for further. use.. . 32 . 3.3.2 Preparation of Fe3O4@Au Nanoparticles. Fe3O4 core/Au shell nanoapeticles were synthesized by deposition of Au on the. performed Fe3O4 nanoaprticles using a modification of Lyon’s iterative. hydroxylamine seeding procedure.[61] First, 1 mL of Fe3O4 nanoparticles solution was. stirred with 1 mL of 0.1 M sodium citrate for 10 min. Next, the solution was diluted. with DI water to 20 mL, and 100 μL of 80 mM NH2OH·HCl solution was added.. Then 2 mL of 1% HAuCl4 solution was added dropwise with stirring. Finally, the. uncoated Fe3O4 nanoparticles were removed by centrifugation.. 3.3.3 DNA Hybridization. The boughten oligonucleotides, which look like white powder, were dissolved in. TE buffer (10 mM Tris-HCl/1 mM EDTA)/50 mM NaCl with pipetting to form. ssDNA solution. A15T3 dsDNA was hybridized by mixing A15 ssDNA and T3. ssDNA sequences at a 1: 1 molar ratio, heating the solution to 95oC for 10 min in a. large water bath, and then cooling slowly to room temperature. The obtained dsDNA. was stored in 4oC.. 3.3.4 Fe3O4@AuNPs-Bound dsDNA. For preparation of drug carrier, the dsDNA were added into Fe3O4@AuNP. solution. The drug carrier was made by mixing 1 mL of 0.1mg/mL Fe3O4@AuNP. solution and 10 μL of 50 mM dsDNA for 6 h at room temperature. To remove. unbound dsDNA, the solution was centrifuged, and the pellet was washed with DI. water twice, and then resuspended in 1 mL of DI water (Figure 3.2).. . 33 . Figure 3.2 Schematic steps for the preparation of Fe3O4@AuNPs-bound dsDNA.. 3.3.5 Doxorubicin-Intercalated Fe3O4@AuNPs-dsDNA. For drug loading, 1 mL of 0.1mg/mL Fe3O4@AuNP-dsDNA was mixing with. 5μL of 1.72 mM doxorubicin for 3 h at room temperature. The unintercalated. doxocrubicin were removed by centrifugation, and the pellet was resuspended in 1. mL of DI water (Figure 3.3).. Figure 3.3 Schematic steps for the preparation of doxorubicin-intercalated. Fe3O4@AuNPs-dsDNA.. . 34 . 3.3.6 Drug Delivery of Fe3O4@AuNPs-dsDNA-Doxorbuicin at. Various Temperatures. For testing the drug release as a function of temperature, each 1 mL of. Fe3O4@AuNP-dsDNA-doxocrubicin was put into water bath with different. temperature for 10 min, and then the sample was immediately removed and. centrifuged to collect the supernatant, which contained the released doxorubicin. To. determine the release rate, the fluorescence intensity of the supernatant was measured.. 3.3.7 Drug Delivery of Fe3O4@AuNPs-dsDNA-Doxorbuicin under. High Frequency Magnetic Field. For inducing drug release by high frequency magnetic fields, 1 mL of. Fe3O4@AuNP-dsDNA-doxocrubicin was put in the center of the loop of high. frequency magnetic field without any contact for different time periods (Figure 3.4).. After treating with high frequency magnetic field, the sample was immediately. removed and centrifuged to collect the supernatant, which contained the released. doxorubicin. By measuring the fluorescence intensity of the supernatant, the release. rate induced by high frequency magnetic field was determined.. . 35 . Figure 3.4 (a) Equipment of drug release induced by high frequency magnetic field.. (b) The sample was put in the center of the loop without any contact.. . 36 . Chapter 4: Results and Discussion. 4.1 Synthesis and Characterization of Fe3O4 Nanoparticles and. Fe3O4@Au Nanoparticles. This section describes the results of Fe3O4 nanoparticles and Fe3O4@Au. nanoparticles with SEM, TEM, EDS, XRD, SAED, zeta-potential, and magnetic. properties analysis.. 4.1.1 Synthesis of Fe3O4 Nanoparticles and Fe3O4@Au Nanoparticles. At first step, we synthesized Fe3O4 naniparticles by co-precipitation. The. particles showed a black color, and can be isolated by a magnet (Figure 4.1).. Although the newly synthesized particles presented a serious aggregation, which were. common in co-precipitation,[16] 0.01 M tetramethylammonium hydroxide (TMAOH). was introduced to the solution for improving this phenomenon. Dramatically, the. particles showed more dispersed. By increasing the concentration of TMAOH from. 0.01 M to 0.1 M, the particles exhibited a more monodispersed morphology. Further,. dispersing the particles in 0.1 M TMAOH with sonication, the isolated Fe3O4. nanoparticles were appeared (Figure 4.2). The improvement may be contributed to the. existence of TMAOH, which is a strong alkaline. The effect of TMAOH will discuss. in 4.1.2. With monodispersed morphology, Fe3O4 nanoparticles had good. performances in MR imaging for medical diagnosis, controlled drug delivery,. biological targeting, and catalysis.. . 37 . Figure 4.1 (a) Fe3O4 nanoparticles synthesized by co-precipitation showed a black. color, and (b) were concentrated by a magnet.. Figure 4.2 SEM images of Fe3O4 nanoparticles in different solvents: (a) DI water, (b). 0.01 M TMAOH, (c) 0.1 M TMAOH, (d) 0.1 M TMAOH with sonication.. . 38 . Since Fe3O4 nanoparticles, which had a large ratio of surface-area to volume,. tend to agglomerate in order to reduce their surface energy by strong magnetic. dipole-dipole attractions between particles. Au shell was covered to enhance chemical. stability for protecting the magnetic core from oxidation and corrosion, as well as. exhibit good biocompatibility and affinity via thiol/amine terminal groups.. After the process of coating Au shell, the particles which can still be isolated by. a magnet, exhibited a pink color (Figure 4.3), with the particle size about 40 nm. The. UV-Vis spectrum also showed a strong Surface Plasmon Resonance (SPR) at ca. 530. nm, which was not shown on the Fe3O4 nanoparticles (Figure 4.4). Among pure Au. nanoparticles, the collective oscillations of free electrons, known as Surface Plasmon. (SP), cause an absorption peak to appear in the visible region of the electromagnetic. spectrum.[62] Factors that affect the position of the SP peak have been investigated. on the basis of Mie theory; for Au nanoparticles, the SP has been shown to shift as a. function of particle size, and stabilizing ligand.[63-68] Therefore, the bimetallic. core/shell nanoparticles were further characterized by UV-Vis spectroscopy to. compare the optical properties to those of pure Au nanoparticles.[61] Au nanoparticles,. which exhibit a 530 nm absorption, have corresponsive 48 nm particle size.[69]. However, the particles prepared in this work exhibited 530 nm absorption with the. size of 40 nm. This phenomenon could result from the charge variation of the Au. particles within the core/shell structure.[70]. . 39 . Figure 4.3 (a) Au shell processed Fe3O4 nanoparticles showed a pink color, and (b). were concentrated by a magnet.. Figure 4.4 (a) SEM image of the Fe3O4 nanoparticles after coating Au shell. (b). UV-Vis spectrum of Fe3O4 nanoparticles (dash) and Fe3O4 nanoparticles after coating. Au shell (solid).. 4.1.2 Zeta-potential of Fe3O4 Nanoparticles and Fe3O4@Au. Nanoparticles. Zeta-potential is a value typically for quantifying the charge distribution, which. represents the coupled effect of the conjugate’s charge and the ionic conditions of the. . 40 . solution. Thus, the changes of particle surface due to the addition of different. surfactants can be figured out by zeta-potential measurement. As shown in Table 4.1,. the zeta-potential of Fe3O4 nanoparticles in DI water, 0.01 M TMAOH, and 0.1 M. TMAOH were -38.84 mV, -40.84 mV, and -43.61 mV respectively; it was showed. that the most negative value of Fe3O4 nanoparticles in 0.1 M TMAOH. It is well. known that TMAOH is a strong alkaline, which can make the surface of the Fe3O4. nanoparticles more negative charged in stronger alkaline medium. Therefore, with. increased amount of TMAOH, zeta-potential of the nanoparticles becomes more. negative.[71] Further, after coating Au shell, the zeta-potential value increasing to. -52.7 mV, showed the most negative charge of Fe3O4@Au nanoparticles.. Sample Zeta-potential. Fe3O4 nanoparticles in DI water -38.84 mV. Fe3O4 nanoparticles in 0.01 M TMAOH -40.84 mV. Fe3O4 nanoparticles in 0.1 M TMAOH -43.61 mV. Fe3O4@Au nanoparticles -52.7 mV. Table 4.1 Zeta-potential of Fe3O4 nanoparticles in DI water, 0.01 M TMAOH, 0.1 M. THAOH, and Fe3O4@Au nanoparticles.. 4.1.3 Size Distribution of Fe3O4 Nanoparticles and Fe3O4@Au. Nanoparticles. With the Au shell we have capped, the difference of particle size between Fe3O4. and Fe3O4@Au could be an evidence for existence of Au shell. For this purpose, the. size distribution of Fe3O4 nanoparticles and Fe3O4@Au nanoparticles were measured.. . 41 . The particle size of the Fe3O4 nanoparticles and the Fe3O4@Au nanoparticles were. calculated by the software named Image-pro plus (IPP), and the analysis region were. the full SEM images of Figure 4.2 (d) and Figure 4.3 (a). The average size of the. Fe3O4 nanoparticles and the Fe3O4@Au nanoaprticles were 9.6±3 nm and 38.1±5.5. nm (Figure 4.5). In other words, the thickness of Au shell wrapped on Fe3O4 could be. calculated as about 14.25 nm with narrow size distribution. Besides, particle size is of. great importance in design of nanomaterials for targeted drug delivery. Particles larger. than 150 nm could be removed rapidly by liver and spleen after injection, resulting in. poor in vivo circulation time and targeting efficiency; while particles smaller than 20. nm have been reported to be excreted through renal clearance, which will also. decrease the longevity of the particles after injection in vivo.[72-73] Thus, the particle. size of Fe3O4@Au nanoparticles prepared in this work fell into the suitable range.. Figure 4.5 Size distributions of (a) Fe3O4 nanoparticles, (b) Fe3O4@Au nanoparticles.. 4.1.4 Characterization of Fe3O4 Nanoparticles and Fe3O4@Au. Nanoparticles. Although all the SEM images indicated the existence of Au shell, TEM images,. EDS spectrum, XRD spectra, and SAED patterns were used to further investigate the. compositions and structures of Fe3O4 nanoparticles and Fe3O4@Au nanoparticles.. . 42 . From the TEM images (Figure 4.6), the color differences of outer and inner. particles showed a core/shell morphology, and all the particles had a uniform size. about 40 nm without any aggregation, which corresponded to SEM image.. Furthermore, both the SEM and TEM images did not show any Fe3O4 nanoparticles,. illustrating that centrifugation to remove uncoated Fe3O4 nanoparticles was effective.. In addition, to confirm the composition of the particles, EDS spectrum were collected. during TEM imaging. This spectrum (Figure 4.7) confirmed the presence of both Fe. and Au elements in the Fe3O4@Au nanoparticles. Cu peaks were caused by scattering. of the Cu TEM grid.. Figure 4.6 (a) TEM image of the Fe3O4 nanoparticles processed coating Au shell. (b). The particles exhibited core/shell morphology.. . 43 . Figure 4.7 EDS spectrum of the sample used to obtain Figure 4.6.. XRD spectrum was used to verify crystal structure and atomic spacing by. constructive interference of monochromatic X-rays and a crystalline sample. The. XRD pattern of Fe3O4 nanoparticles showed diffraction peaks at 21.16°, 30.11°, 35.53°,. 43.12°, and 62.61° which can be indexed to the (002), (112), (103), (004), and (224). plans of Fe3O4 in a cubic phase with the standard pattern of the Joint Committee of. Power Diffraction Standard (JCPDS) code of 75-1609. The XRD pattern of. Fe3O4@Au nanoparticles showed diffraction peaks at 38.18°, 44.38°, 64.57°, and. 77.56° which can be indexed to the (111), (200), (220), and (311) plans of Au in a. cubic phase with JCPDS code of 65-2870. The XRD data showed the peaks of. Fe3O4@Au nanoparticles correspond to gold (Au) but magnetite phase (Fe3O4). This. phenomenon may be contributed to the high ratio of Au to Fe3O4 (Figure 4.8). The. results indicated that the prepared Fe3O4 nanoparticles can be confirmed as. composition of Fe3O4, and the prepared Fe3O4@Au nanoparticles can prove the. existence of Au.. . 44 . Figure 4.8 XRD spectra of (a) Fe3O4 nanoparticles, and (b) Fe3O4@Au nanoparticles.. The selected-area electron diffraction (SAED) pattern was employed to further. study the core/shell structure of the nanoparticles. The SAED pattern of Fe3O4. nanoparticles can be indexed as Fe3O4 (200), (230) and (133) with JCPDS code of. 89-6466, and the SAED pattern of Fe3O4@Au nanoparticles can be indexed as Fe3O4. (122) and (006) with JCPDS code of 89-6466 and Au (311) and (220) with JCPDS. code of 89-3697 and 65-2870 (Figure 4.9). Both SAED patterns and TEM images. proved the core/shell structure of the Fe3O4@Au nanoparticles with the same. compositions indicated by XRD patterns.. . 45 . Figure 4.9 SAED patterns of (a) Fe3O4 nanoparticles, and (b) Fe3O4@Au. nanoparticles..