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Nanoparticle-mediated Cell Behavior Modulation for the Treatment of Myocardial Infarction : 나노입자 기반, 세포 거동조절을 통한

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dc.contributor.advisor김병수-
dc.contributor.author한진-
dc.date.accessioned2017-10-27T16:47:19Z-
dc.date.available2017-10-27T16:47:19Z-
dc.date.issued2017-08-
dc.identifier.other000000144961-
dc.identifier.urihttps://hdl.handle.net/10371/136866-
dc.description학위논문 (박사)-- 서울대학교 대학원 공과대학 화학생물공학부, 2017. 8. 김병수.-
dc.description.abstractMyocardial infarction (MI) is one of the leading causes of death worldwide, and accounts for majority of cardiac-associated disorders. MI originates from reduced blood supply to the heart and subsequent cardiac necrosis, hence, tissue engineering approaches are required for successful cardiac repair. Recently, various types of cells and nanoparticles drew significant attention as efficient therapeutics for cardiac repair, and combinatorial treatments between cells and nanoparticles have been introduced. Even so, majority of nanomaterials have been mostly utilized as delivery carriers, and studies regarding how nanoparticles actively modulate cell behaviors and potentiate the therapeutic efficacy of these cells remain unexplored.
Current dissertation presents the integration of stem or immune cells with most widely used nanoparticles, such as iron oxide nanoparticles or graphene oxide, for the treatment of MI. More specifically, biological role of these nanoparticles and how innate chemical properties of nanoparticles mediate cell behaviors is elucidated. Major goals of dissertation are summarized as follows
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dc.description.abstract1) Elucidation of metal ion-delivering capability of iron oxide nanoparticles, and investigation on the modulation of cell signaling transduction and development of intercellular gap junction crosstalk 2) Elucidation of sp2 chemistry-based intracellular antioxidant chemistry of graphene oxide, and its immune modulatory function for therapeutic polarization of macrophages in MI treatment.
First, we showed that iron oxide nanoparticles can modulate intracellular signaling transduction in cardiac cells, and improve intercellular gap junction formation in stem cell co-culture. Co-culture of stem cells with cardiac cells has windowed a platform for cardiac priming of MSCs prior to in vivo transplantation, and active gap junctional crosstalk between stem cells and cardiac cells are crucial in stem cell modification. In this study, we report that iron oxide nanoparticles can augment the expression of gap junction protein connexin 43 in cardiac cells to better form gap junction channels with stem cells. Stem cells co-cultured with nanoparticle-harboring cardiac cells exhibited active biomolecule transfer and showed increased level of electrophysiological cardiac biomarkers and cardiac repair-favorable paracrine secretion. Implanted in rat MI models, cardiac-primed stem cells significantly reduced cardiac fibrosis, promoted cardiac tissue regeneration and function.
Secondly, we exhibited that graphene oxide with carbon-based sp2 chemistry can function as reactive oxygen species scavenger within the cells and prevent inflammatory activation of macrophages. Furthermore, we functionalized graphene oxide nanoparticles with plasmid DNA to better polarize inflammatory cells at cardiac infarction area into tissue regenerative macrophages. After the onset of MI, excessive amount of inflammatory macrophages propagates at the peri-infarct to exacerbate tissue necrosis, suggesting that uncontrolled differentiation and activation of inflammatory macrophages greatly hamper proper tissue regeneration. In this study, we demonstrated that graphene oxides can act as an antioxidant to prevent inflammatory activation of macrophages, and further DNA functionalization significantly improved therapeutic polarization of these macrophages. Furthermore, injection of DNA-functionalized graphene oxides in mouse MI models notably reduced immune cell infiltration and mitigated cardiac fibrosis for cardiac performance improvement.
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dc.description.tableofcontentsChapter 1. Research backgrounds and objectives 1
1.1. Myocardial infarction (MI) and current therapeutics 2
1.2. Cell therapy for MI 5
1.2.1. MSC-mediated therapy for MI 5
1.2.2. Macrophage therapy for MI 9
1.3. Nanomaterial-mediated cell therapy for MI 12
1.3.1. Nanomaterial-mediated stem cell delivery 14
1.3.2. Topographical cues of nanomaterials for stem cell and macrophage behavior modulation 16
1.3.3. Electrical properties of nanomaterials for stem cell behavior modulation 18
1.3.4. Intrinsic properties of nanomaterials for stem cell and macrophage behavior control 20
1.4. Limitations of previous cell or nanomaterial-mediated therapy 21
1.5. Iron oxide nanoparticles (IONPs) and graphene oxide (GO) for cell modulation and tissue engineering 22
1.6. Research objectives 23
Chapter 2. Experimental procedures 24
2.1. Preparation of iron oxide nanoparticle (IONP) 25
2.2. Characterization of IONP 26
2.3. Cell preparation and IONP-based cell culture 27
2.3.1. MSC and H9C2 culture using IONP and nanoparticle toxicity 27
2.3.2. IONP-based MSC co-culture 28
2.3.3. IONP-mediated cell sorting after co-culture 29
2.4. In vitro analysis 30
2.4.1. TEM analysis 30
2.4.2. Fluorescent images 31
2.4.3. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) 32
2.4.4. Western blot assay 33
2.4.5. Calcein-AM dye transfer assay 34
2.4.6. Immunocytochemistry 35
2.4.7. Paracrine profile analysis 36
2.5. Rat MI model and cMSC treatment 37
2.6. In vivo assessment 38
2.6.1. Histological and immunohistochemical assessment 38
2.6.2. Evaluation of cardiac performance 39
2.7. Preparation of graphene oxide (GO) and GO derivatives 40
2.8. Characterization and DNA conjugation of GO derivatives 42
2.9. Cell preparation and uptake of GO derivatives 43
2.9.1. Culture of mouse bone marrow-derived macrophages 43
2.9.2. Uptake and cellular affinity of GO derivatives 44
2.10. In vitro analysis 45
2.10.1. Metal chelating assay 45
2.10.2. Intracellular reactive oxygen species (ROS) generation 46
2.10.3. qRT-PCR and western blot assay 47
2.10.4. Paracrine secretion analysis 48
2.10.5. Fluorescent imaging 49
2.10.6. Co-culture of macrophages and cardiomyocytes 50
2.11. Mouse MI model and MGC injection 51
2.12. In vivo assessment 52
2.12.1. Histological assessment and evaluation of genes and proteins 52
2.12.2. Evaluation of cardiac performance 54
2.13. Statistical analysis 55
Chapter 3. Iron oxide nanoparticle-mediated development of cellular gap junction crosstalk to improve mesenchymal stem cells therapeutic efficacy for myocardial infarction 56
3.1. Introduction 57
3.2. Results and discussion 61
3.2.1. Internalization of IONP and H9C2 behavior modulation 61
3.2.2. IONP-based magnetic H9C2 sorting post co-culture 66
3.2.3. Cardiac phenotype development in MSCs after co-culture 69
3.2.4. Cardiac repair-favorable paracrine profile in MSCs after co-culture 72
3.2.5. Attenuation of left ventricular remodeling 75
3.2.6. Improved vessel density 77
3.2.7. Enhancement in animal survival and cardiac function 79
Chapter 4. Intracellular antioxidant function development via DNA-functionalized graphene oxide to modulate inflammation and repolarize macrophages for the treatment of myocardial infarction 82
4.1. Introduction 83
4.2. Results and discussion 88
4.2.1. Preparation and characterization of macrophage-targeting/polarizing graphene oxide complex (MGC) 88
4.2.2. Selective cellular uptake and cytotoxicity of MGC 91
4.2.3. Reactive oxygen species scavenging and inflammation modulation by MGC 95
4.2.4. Polarization of M1 macrophages to M2 macrophages 99
4.2.5. Attenuation of inflammation and early shift to reparative M2 phase after MGC/IL-4 pDNA injection in vivo 106
4.2.6. Improved left ventricular remodeling and increased vessel density in vivo 111
4.2.7. Improved recovery of cardiac function 116
Chapter 5. Conclusions 119
References 122
요약 (국문 초록) 156
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dc.formatapplication/pdf-
dc.format.extent8303234 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subject산화철 나노입자-
dc.subject그래핀 옥사이드-
dc.subject세포 거동조절-
dc.subject심근경색-
dc.subject조직공학-
dc.subject.ddc660.6-
dc.titleNanoparticle-mediated Cell Behavior Modulation for the Treatment of Myocardial Infarction-
dc.title.alternative나노입자 기반, 세포 거동조절을 통한-
dc.typeThesis-
dc.description.degreeDoctor-
dc.contributor.affiliation공과대학 화학생물공학부-
dc.date.awarded2017-08-
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