암 치료진단용 제어합성 및 산화철 나노입자 기반의 표면 처리 = Controlled Synthesis and Surface Tailoring of Iron Oxide Nanoparticle Based Nanocomposites for Cancer Theranostics

국문 초록 (Abstract)

나노기술은 나노의학에서 최근 많은 관심과 급격한 발전을 보이고 있는 분야이다. 나노기술 기반의 치료법들의 특수성으로 인해 다양한 분야의 과학자들의 관심을 받고 있으며 그 중에서도 특히 암치료진단학 분야에서 주목을 받고 있다. 최근의 암치료에 관한 연구동향은 암의 이른 진단 및 최소한의 부작용을 가진 효과적인 치료법을 개발하는 것이다. 이러한 연구적 관심에 있어서 생체친화적 자성 나노입자(MNPs)는 MRI, 세포 및 조직들의 표적화, 약물전달, 온열치료 등에 사용될 수 있는 다양한 물성들로 인해 암 치료진단학에서 매우 중요한 역할을 할 것으로 여겨진다. 최근 생체친화적 자성 나노입자의 온열치료에 이용될 수 있는 교번자기장 내 발열성, 암세포 특이적인 약물전달 성질, 또한 물분자의 자기장내 양자방출을 가속화시키는 성질을 이용한 MRI T2 영상에서의 조영제로서의 역할 등의 다양한 기능적 특성과 그 상승작용으로 인해 인해 암진단학에서의 사용을 위한 이 물질의 관심이 증폭되고 있다. 그러므로 본 연구는 자성 나노입자와 약물들을 이용하여 MRI 효율성을 증대시키며, 단일 온열치료나 항암화학치료 기반의 기존 일반적인 치료법들보다 우월한 치료 효율을 보이는 다기능성 치료적 나노시스템 개발을 목적으로 한다.
본 연구는 독특하게 기능화된 자성나노입자와 나노섬유들을 포함한 상승작용적 암치료를 위해 다양한 나노합성들을 제시한다. 이 연구의 주된 목적은 자극반응성 약물 전달과 MRI 영상의 향상 그리고 일부의 온열치료적효과를 동시에 가능하게 하는 스마트 기능화 전략 설계에 있다. 암특이적 항암 약물전달을 위해 암세포 주변의 산성 환경에서 낮은 pH에 반응하여 약물이 방출되는 pH반응성 약물전달체계가 이용된다. 홍합 추출물의 카테콜 그룹들의 독특한 특성을 이용한 poly (2-Hydroxyethyl methacrylate-co-dopamine methacrylamide) p(HEMA-co-DMA) and poly (methyl methacrylate-co-dopamine methacrylamide) p(MMA-co-DMA)와 같은 혼성 중합체들을 합성하여 강자성 산화철 나노입자들과 자성 나노섬유들의 표면기능화에 사용하였다. 이 상승작용적 항암치료법은 붕산을 포함하고 있는 bortezomib(BTZ)을 앞서 말한 폴리머들의 카테콜기들에 중합시켜 제작되었다.홍합 유래물을 이용한 스마트 자성 나노섬유는 또한 현저한 MRI 영상 향상을 통해 추가적인 항암치료적 상승효과를 보였다.
또한 연구의 일부로 암세포 특이적 약물 방출 및 내시경적 온열치료법에 사용가능한 체내 삽입용 스마트 자성 나노섬유 장치를 제작하여 in vitro 실험이 완료되었다. 이 IONPs를 위해서, 생체친화적, 생체재흡수성폴리머인 poly(d,l-lactide-co-glycolide) (PLGA)를 전기방사법을 이용하여 나노섬유매트를 제작하였고, 암특이적 약물전달을 위해 BTZ와 카테콜의 pH반응성 결합을 이용했다. 독창적인 강자성 중심부-외피 형태의 망간 철 나노입자(MFNPs) 는 다공성 이산화규소 나노입자들에 쌓여(MSMFNPs) 항암제인 독소루비신(DOX)과 결합시켜 온열치료 및 항암치료의 병용치료로 사용되기 위해 개발되어 그 항암치료의 효과를 체외 실험을 통해 실험되었다. MSMFNPs를 통해 잠재적으로 양이온성 약물전달에 이용하기 위해 이 나노입자는 풍부한 카복실기와 하이드록실 그룹을 가지며 pH반응성을 보이는 1, 2-cyclohexanedicarboxylic anhydride (CDA)를 클릭링커로 이용했다. 산화 그라핀-산화철-독소루비신(GO-IO-DOX)을 이용한 암진단치료적인 플랫폼 또한 제작되어 사용되었고, 이 스마트 자성 나노플랫폼이 교번자기장내에서 열전달을 하는 온열치료적 제제로서와 암특이적으로 pH반응성 약물 전달기능을 통한 항암제제, 또한 T2 조영제로서 MRI 영상을 향상 시키는 기능을 동시에 해낼 수 있음이 보였다. 결론적으로, 근래의 연구들은 성공적으로 항암치료 및 진단을 동시에 수행할 수 있는 독창적인 나노합성법들을 개발하는 것에 성공했다.

다국어 초록 (Multilingual Abstract)

Nanotechnology is rapidly advancing with burgeoning interest in the field of Nanomedicine. The uniqueness of Nanotechnology-based therapeutics has been attracted scientists from various research areas especially in the field of cancer theranostics. The frontiers of cancer research include the early detection of cancer as well as the cancer treatment with few side effects. Therefore in this regard biocompatible magnetic nanoparticles (MNPs) play an important role in cancer theranostics by combining its various properties include magnetic resonance imaging (MRI), cell and tissue targeting, drug delivery and hyperthermia. Recently there has been a critical thrust aroused towards a synergistic cancer theranostics by combining the unique heat generation property of the magnetic nanoparticles in an alternating magnetic field (AMF) to induce hyperthermia along with the exclusive drug delivery properties specific for cancer cells as well as the enhanced ability of magnetic nanoparticles to accelerate the MRI relaxation process of the surrounding water protons as a T2 contrast agents for MRI. Therefore the present research aims to develop multifunctional therapeutic nanosystems incorporating both magnetic nanoparticles and drugs, and apply their superior efficacy in treating cancer compared to either hyperthermia or chemotherapy as standalone therapies along with MRI performance.
The research put forward different nanoformulations for the synergistic cancer therapy including uniquely functionalised magnetic nanoparticles and nanofibers. Main focus of the research include the design of smart functionalization strategies for the magnetic nanoparticles to enable stimuli-responsive drug delivery along with mild hyperthermia and enhanced MRI response. For the tumor specific anticancer drug delivery, pH responsive drug delivery system is mainly employed by benefiting the slightly acidic extracellular environment of tumor tissues, which allow for pH-triggered release of the anticancer drugs. The unique properties of mussel-inspired multiple catecholic groups –presenting unique copolymers such as poly (2-Hydroxyethyl methacrylate-co-dopamine methacrylamide) p(HEMA-co-DMA) and poly (methyl methacrylate-co-dopamine methacrylamide) p(MMA-co-DMA) are synthesized and exploited to surface functionalize the superparamagnetic iron oxide nanoparticles and fabricate magnetic nanofibers respectively. The synergistic anticancer therapy application is made possible by conjugating the borate containing anticancer drug Bortezomib (BTZ) to the catechol moieties presented by the formers. Besides the synergistic anticancer efficacy, the mussel inspired smart magnetic nanofibers also exhibits remarkable MRI property.
An implantable smart magnetic nanofiber device for endoscopic hyperthermia treatment and tumor triggered controlled drug release is also fabricated and tested in vitro as part of the research. For this IONPs incorporated nanofiber matrix were developed by electrospinning the biocompatible and bioresorbable polymer poly(d,l-lactide-co-glycolide) (PLGA) and the tumor triggered anticancer drug delivery is realized by the pH sensitive binding of catechol and BTZ. A novel superparamagnetic core–shell manganese ferrite nanoparticles (MFNPs) encapsulated mesoporous silica nanoparticles (MSMFNPs) loaded with anticancer drug doxorubicin (DOX) for the combined application of hyperthermia and chemotherapy was also developed and tested its antitumor efficacy in vitro. Inorder to exploit the MSMFNPs, for the potential applications of cationic drug delivery, the nanoparticles are modified by a click linker, 1, 2-cyclohexanedicarboxylic anhydride (CDA), possessing an abundance of carboxyl and hydroxyl groups, exhibiting pH-sensitivity. A theranostic cancer platform using Graphene Oxide–Iron Oxide-Doxorubicin (GO-IO-DOX) is also fabricated to apply for its efficacy in cancer theranostics and found that the smart magnetic nanoplatform acts both as a hyperthermic agent that delivers heat when an alternating magnetic field is applied and a chemotherapeutic agent in a cancer environment by providing a pH-dependent drug release to administer a synergistic anticancer treatment with an enhanced T2 contrast for MRI. Thus in conclusion, the current research succeeded in developing unique nanoformulations for the synergistic anticancer treatment and diagnosis.

목차 (Table of Contents)

• Chapter 1.Introduction 1
• 1.1 Background 1
• 1.2 Thesis overview 3
• 1.3 Physics of magnetism 7
• 1.3.1 Classification of magnetic materials 7
• 1.3.2 Curie Temperature 8
• 1.3.3 Superparamagnetism 9
• 1.4 Heat dissipation mechanism of MNP 12
• 1.4.1 Specific Absorption Rate (SAR) 15
• 1.5 Hyperthermia 16
• 1.5.1 Methods of Hyperthermia 17
• 1.5.2 Mechanism of Hyperthermia 18
• 1.5.3 Tumor selective effect of hyperthermia 18
• 1.5.4 Magnetic nanoparticle Hyperthermia 20
• 1.5.5 Administration of MNPs to the tumor site 21
• 1.5.6 Advantages of MNP hyperthermia 24
• 1.5.7 Magnetic nanomaterials used for hyperthermia 27
• 1.6 Superparamagnetic iron oxide nanoparticle (SPION) 28
• 1.6.1 Synthesis of SPION 30
• 1.6.2 SPION as Hyperthermia agent 33
• 1.6.3 SPION as MR contrast agent 34
• 1.6.4 SPION as drug delivery carrier 39
• 1.7 Synergistic effect of combination therapy 43
• 1.8 Magnetic Nanosystems for thermo-chemo therapy 44
• 1.8.1 Different nanosystem designs 45
• 1.8.1.1 Liposomes 45
• 1.8.1.2 Micelle 46
• 1.8.1.3 Polymeric nanoformulations 47
• 1.8.1.4 Core/shell nanoparticles 48
• 1.8.1.5 Magnetic nanofiber 49
• 1.9 Multifunctional MNPs for cancer theranostics 50
• 1.10 Objective of the research 51
• Chapter 2. A smart magnetic nanoplatform for synergistic anticancer therapy: manoeuvring mussel-inspired functional magnetic nanoparticles for pH responsive anticancer drug delivery and Hyperthermia 54
• Abstract 54
• 2.1 Introduction 56
• 2.2 Experimental Section 59
• 2.2.1 Materials 59
• 2.2.2 Synthesis of Iron oxide nanoparticles (IONPs) 59
• 2.2.3 Synthesis of dopamine methacrylamide (DMA) 60
• 2.2.4 Synthesis and Characterization of p(HEMA-coDMA), abbreviated as HEDO 60
• 2.2.5 Synthesis of Iron oxide nanoparticles functionalized with HEDO, abbreviated as HEDO-Fe3O4 61
• 2.2.6 Preparation of the HEDO-Fe3O4-BTZ nanoparticles 61
• 2.2.7 Characterization of the nanoparticles 62
• 2.2.8 In vitro Hyperthermia studies of HEDO-Fe3O4 and SAR measurement 63
• 2.2.9 Kinetics of the pH-dependent drug release 65
• 2.2.10 In vitro cell culture studies 65
• 2.2.11 Biocompatibility study of HEDO-Fe3O4 65
• 2.2.12 Intracellular localization study of HEDO-Fe3O4 66
• 2.2.13 In vitro hyperthermia study using HEDO-Fe3O4-BTZ 67
• 2.2.14 Live/dead assay 68
• 2.2.15 In vivo tumor inhibition study using HEDO-Fe3O4- BTZ 69
• 2.3 Results and Discussion 70
• 2.4 Conclusions 88
• Chapter 3. An implantable smart magnetic nanofiber device for endoscopic hyperthermia treatment and tumor-triggered controlled drug release 90
• Abstract 90
• 3.1 Introduction 91
• 3.2 Experimental Section 94
• 3.2.1 Materials 94
• 3.2.2 Synthesis of Iron Oxide Nanoparticles (IONPs) 95
• 3.2.3 Synthesis of magnetic nanofiber(MNF) 95
• 3.2.4 Synthesis of mussel-inspired magnetic nanofibers (MMNF) 96
• 3.2.5 Synthesis of bortezomib-loaded mussel inspired magnetic nanofibers (BTZ-MMNF) 97
• 3.2.6 Characterization of IONPs, MNF, MMNF 97
• 3.2.7 AMF-Induced Heating ability of MMNF and IONPs 98
• 3.2.8 Drug release from the MMNF-BTZ nanofiber 99
• 3.2.9 In vitro cell culture studies 100
• 3.2.10 Biocompatibility study of MMNF 100
• 3.2.11 In vitro anticancer studies 100
• 3.2.12 Qualitative analysis 102
• 3.2.13 Statistical analysis 103
• 3.3 Results and Discussion 103
• 3.4 Conclusion 121
• Chapter 4. Mussel-Inspired Electrospun Smart Magnetic Nanofibers for Hyperthermic Chemotherapy 122
• Abstract 122
• 4.1 Introduction 123
• 4.2 Experimental Section 125
• 4.2.1 Materials 125
• 4.2.2 Synthesis of dopamine methacrylamide (DMA) 126
• 4.2.3 Synthesis and characterization of p(MMA-co-DMA) abbreviated as MADO 126
• 4.2.4 Synthesis of Iron oxide nanoparticles (IONPs) 127
• 4.2.5 Fabrication of catecholic nanofibers MADO 127
• 4.2.6 Fabrication of drug bound Catecholic Nanofibers MADO-BTZ 128
• 4.2.7 Fabrication of catecholic magnetic nanofibers MADO-Fe3O4 and drug bound catecholic magnetic nanofibers MADO-Fe3O4-BTZ 128
• 4.2.8 Sample preparation for boron NMR spectroscopy 129
• 4.2.9 Characterization of IONPs and MADO nanofibers 129
• 4.2.10 AMF-Induced Heat Generation Properties and calculation of SAR 130
• 4.2.11 Biocompatibility study of nanofiber 132
• 4.2.12 Drug release from MADO-BTZ nanofiber 132
• 4.2.13 In vitro anticancer study 133
• 4.2.14 Live/dead assay 134
• 4.3 Results and Discussion 135
• 4.4 Conclusion 154
• Chapter 5. Design and application of a smart nanodevice by combining cationic drug delivery and hyperthermia for cancer apoptosis 156
• Abstract 156
• 5.1 Introduction 157
• 5.2 Experimental Section 160
• 5.2.1 Materials 160
• 5.2.2 Synthesis of manganese ferrite nanoparticles (MFNPs) 160
• 5.2.3 Synthesis of mesoporous core-shell-MF-silica nanoparticles (MSMFNPs) 161
• 5.2.4 Synthesis of DOX-loaded mesoporous core-shellMF-silica nanoparticles (DOX MSMFNPs) 162
• 5.2.5 Characterization of techniques 163
• 5.2.6 In vitro hyperthermia studies and SAR measurements 164
• 5.2.7 Kinetics of pH-dependent drug release 165
• 5.2.8 In vitro cell culture studies 165
• 5.2.9 Intracellular localization of MSMFNPs by 4T1 cell lines 165
• 5.2.10 In vitro biocompatibility of MSMFNPs 166
• 5.2.11 In vitro anticancer studies 167
• 5.3 Results and Discussion 168
• 5.3 Conclusion 183
• Chapter 6. Multifunctional Nanocarpets for Cancer Theranostics: Remotely Controlled Graphene Nanoheaters for ThermoChemosensitisation and Magnetic Resonance Imaging 184
• Abstract 184
• 6.1 Introduction 185
• 6.2 Experimental Section 188
• 6.2.1 Materials 188
• 6.2.2 Preparation of Graphene Oxide 188
• 6.2.3 Preparation of superparamagnetic iron oxide nanoparticles (SPIONs) 189
• 6.2.4 Preparation of amphiphilic graphene oxide Iron oxide nanocomposite (GO-IO) 190
• 6.2.5 Preparation of biocompatible and hydrophilic graphene oxide iron oxide nanocomposite (GO-IOPEG) 191
• 6.2.6 Preparation of drug encapsulated GO-IO (GO-IODOX) 192
• 6.2.7 Material Characterizations 193
• 6.2.8 In vitro Hyperthermia studies and SAR measurements 194
• 6.2.9 Kinetics of the pH-dependent drug release 195
• 6.2.10 Magnetic Resonance Relaxivity Analysis 196
• 6.2.11 In vitro cell culture studies 197
• 6.2.12 In vitro MR Phantom Imaging 197
• 6.2.13 In-Vitro cytocompatibility study 197
• 6.2.14 Intracellular localization study 198
• 6.2.15 In vitro hyperthermia induced chemo sensitization studies 198
• 6.2.16 In vitro hyperthermia study 200
• 6.2.17 Qualitative analysis of localized tumoricidal effect 200
• 6.3 Result and discussion 201
• 6.4 Conclusion 221
• Chapter 7. Conclusion and future perspective 223
• References 225
• Abstract in Korean 256
• List of publication 260
• List of papers presented in conferences 263
• List of patents 265
• Acknowledgements 266