Synthesis and Biomedical Applications of Isotope-Labeled Graphene Quantum Dots = 동위원소 표지 그래핀 양자점의 합성 및 생체의학적 응용

다국어 초록 (Multilingual Abstract)

2. 1. Abstract
Increased calcium influx is associated with mitochondrial dysfunction, leading to podocyte injury and proteinuria. However, the mechanisms underlying transient receptor potential channel 5 (TRPC5)-induced calcium signaling in renal mitochondrial dysfunction remain largely unknown. Therefore, we investigated whether graphene quantum dots (GQDs) can downregulate TRPC5-controlled calcium influx signaling pathways and reverse mitochondrial dysfunction or impaired kidney function. The anti-inflammatory, anti-fibrotic, and anti-apoptotic effects of GQDs were evaluated in vitro using mRNA sequencing, qRT-polymerase chain reaction (PCR), western blotting, immunostaining analysis, Annexin-propidium iodide (PI), senescence, and a wound healing assay. Intracellular calcium signals, mitochondrial function, and morphological changes were also assessed.
In vitro validation indicated that GQDs reduced renal fibrosis in human podocytes under oxidative stress and rotational force-driven pressure by preserving the scaffold architecture of renal cells. GQDs also regulated intracellular calcium signaling and TRPC5 expression and restored mitochondrial function, morphology, energy metabolism, and mitochondrial membrane potential. Moreover, GQDs attenuated fibrosis and apoptosis in fibroblasts and tubular epithelial cells, demonstrating their effectiveness on various renal cells. These results suggest that GQDs are a crucial therapeutic nanomaterial for renal cell injury and modulate calcium-dependent apoptosis associated with mitochondrial injury in the pathophysiology of renal diseases. Thus, GQDs have potential as therapeutic agents for various kidney diseases by slowing the onset and progression of fibrosis.

다국어 초록 (Multilingual Abstract)

3. 1. Abstract
Previous In modern medical imaging, the integration of multiple imaging modalities has become increasingly crucial for comprehensive disease assessment and treatment monitoring. Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET) provide complementary information about physiological and pathological processes. However, both techniques have limitations that can impede their efficacy in certain clinical scenarios. To address these limitations and capitalize on their strengths, the development of dual modal imaging techniques has emerged as a promising approach.
Carbon-13 Graphene Quantum Dots (13C-GQDs) and Copper-64 (64Cu) have gained attention as versatile imaging agents for MRI and PET, respectively. 13C-GQD exhibits favorable biocompatibility and imaging properties suitable for MRI applications, while 64Cu offers excellent PET imaging capabilities due to its favorable decay characteristics.
In this study, we propose integrating 13C-GQDs and 64Cu for dual-modal imaging to enhance their respective strengths in MRI and PET. By combining these agents, our goal is to achieve simultaneous anatomical and functional imaging with improved sensitivity and spatial resolution. We introduce a stable dual-modal imaging agent by conjugating 64Cu to 13C-GQD using a chelator-free approach.

다국어 초록 (Multilingual Abstract)

4. 1. Abstract
This study investigated whether hydroxyapatite (HAp)-mineralized graphene film could support osteogenic differentiation of human adipose-derived, stromal cell (hASCs) in vitro. Graphene was produced by a chemical vapor deposition (CVD) method and the physical and chemical characteristics of the graphene film, which was functionalized with hydroxyapatite mineralization following ultraviolet-ozone (GR_UVO) treatment, were subsequently validated. Results of scanning electron microscopy, X-ray photoelectron spectroscopy and Raman spectroscopy showed GR_UVO for 5 min yielded applicable graphene coverage (97.98 ± 0.85%), conversion of chemical composition ratio (29.78% C-O, 18.34% C=O and 8.49% O-C=O) and degree of oxidation, (I2D /IG ratios 2.22) with maximal density of HAp-graphene layer. In vitro-cell proliferation, viability and adhesion of hASCs after being cultured on HAp-mineralized, graphene-coated glass (HAp/GR) with the optimized GR_UVO treatment (5 min) demonstrated a significant increment of proliferation (1.560.1 vs. 1 to 1.130.1, p<0.05) without changing in viability (94.831% to 95.31.6%, p=0.9651) compared with the control (intact glass). There were no differences in F-actin and Vinculin on day 1 (p=0.1422 and 0.5025, respectively) and on day 4 (p=0.3787 and 0.9208) of culture. Osteogenic differentiation of hASCs was significantly improved on the HAp/GR with increasing of osteogenesis-related genes (Runx2 and Osteocalcin). The hASCs culture with the HAp/GR glass promoted phospho-SMAD1/5/9 and SMAD4 expression with increased patterns of BMP/Smad signal-related genes, regardless of differentiation induction or not. These results demonstrated that hydroxyapatite-mineralized graphene film prepared by CVD method and optimal ultraviolet treatment promoted osteogenic differentiation of hASCs, which BMP/Smad signaling was involved.

다국어 초록 (Multilingual Abstract)

Since the successful exfoliation of graphene in 2004, it has garnered global attention for its exceptional physical, electrical, and chemical properties. In addition, graphene-based materials with controlled sizes and functionalities exhibit unique biological characteristics useful for bioimaging and therapeutic applications. Particularly, graphene quantum dots (GQDs) have been intensively studied for their excellent biocompatibility, capability of reversing the amyloid fibrillation and removing reactive oxygen to suppress inflammation, tunability with various chemical functional groups for targeting or imaging, etc., which are expected to provide further opportunities in the field of nanomedicine for combating incurable diseases.
This thesis delves into the extensive applications of graphene-based nanomaterials, particularly focusing on graphene quantum dots (GQDs). It begins with an overview of graphene and its derivatives, exploring synthesis methods such as Chemical Vapor Deposition (CVD) for graphene and specific techniques for GQD synthesis. The therapeutic potential of GQDs in mitigating renal fibrosis by alleviating oxidative stress and restoring mitochondrial membrane potential is highlighted. Additionally, the development of dual isotope-labelled GQDs for enhanced MRI and PET imaging capabilities, emphasizing their synthesis, characterization, and imaging efficacy, is investigated.
Chapter 1 provides an overview of graphene and its derivatives, detailing various synthesis methods, including Chemical Vapor Deposition (CVD) for graphene and specific methods for graphene quantum dots (GQDs). It explores practical applications of graphene-based nanomaterials, particularly in fibrosis treatment, MRI contrast agents, and tissue engineering. Additionally, it discusses the paramagnetic properties of GQDs as analyzed through Electron Paramagnetic Resonance (EPR).
Chapter 2 presents a study demonstrating the therapeutic potential of GQDs in reducing renal fibrosis. The research highlights how GQDs alleviate oxidative stress and restore mitochondrial membrane potential, leading to significant improvements in kidney function and structure.
Chapter 3 discusses the development and application of dual isotope-labelled GQDs for both MRI and PET imaging. The study showcases the synthesis process, characterization, and dual-modality imaging capabilities, emphasizing the potential of these GQDs in enhancing diagnostic accuracy and imaging efficiency.
Chapter 4 investigates the enhanced osteogenesis of human adipose-derived stromal cells cultured on hydroxyapatite-mineralized graphene films. The findings indicate that this composite material significantly improves cell proliferation and differentiation, making it a promising candidate for bone tissue engineering applications.

국문 초록 (Abstract)

2004년 그래핀의 성공적인 박리 이후, 그래핀은 뛰어난 물리적, 전기적, 화학적 특성으로 전 세계적인 주목을 받았다. 추가로, 크기와 기능이 제어된 그래핀 기반 재료들은 생체 이미징 및 치료 응용에 유용한 독특한 생물학적 특성을 나타낸다. 특히, 그래핀 양자점(GQDs)은 우수한 생체 적합성, 아밀로이드 섬유화 역전 및 반응성 산소 제거를 통해 염증을 억제하는 능력, 타겟팅 또는 이미징을 위한 다양한 화학적 기능 그룹과의 조정 가능성 등으로 집중적으로 연구되었다. 이러한 특성들은 난치병과 싸우기 위한 나노의학 분야에서 더 많은 기회를 제공할 것으로 기대된다.
이 논문은 그래핀 기반 나노재료, 특히 그래핀 퀀텀 닷(GQDs)의 광범위한 응용을 탐구한다. 먼저 그래핀과 그 유도체의 개요를 시작으로, 화학 기상 증착(CVD) 및 GQD 합성 방법을 설명한다. GQD의 신장 섬유증 완화에 대한 치료 잠재력과 산화 스트레스 완화 및 미토콘드리아 막 전위 회복에 대해 강조한다. 또한, MRI 및 PET 이미징을 향상시키기 위한 이중 동위원소 라벨링된 GQD의 개발, 합성, 특성화 및 이미징 효능을 조사한다.
1장에서는 그래핀과 그 유도체의 개요를 제공하며, 화학 기상 증착(CVD) 및 그래핀 퀀텀 닷(GQDs)의 다양한 합성 방법을 상세히 설명한다. 그래핀 기반 나노재료의 실용적인 응용, 특히 섬유증 치료, MRI 조영제, 조직 공학에서의 응용을 탐구한다. 또한, 전자 스핀 공명(EPR) 분석을 통해 GQD의 상자성 특성을 논의한다.
2장에서는 GQD가 신장 섬유증을 줄이는 데 있어 치료 잠재력을 입증하는 연구를 소개한다. 연구는 GQD가 산화 스트레스를 완화하고 미토콘드리아 막 전위를 회복시켜 신장 기능과 구조를 크게 개선하는 방법을 강조한다.
3장은 MRI와 PET 이미징 모두를 위해 이중 동위원소로 라벨링된 GQD의 개발과 응용을 다룬다. 연구는 합성 과정, 특성화, 이중 모드 이미징 능력을 보여주며, 이러한 GQD가 진단 정확도와 이미징 효율성을 향상시킬 수 있는 잠재력을 강조한다.
4장은 하이드록시아파타이트로 미네랄화된 그래핀 필름에서 배양된 인간 지방 유래 간질 세포의 향상된 골 형성을 조사한다. 연구 결과는 이 복합 재료가 세포 증식과 분화를 크게 개선하여 뼈 조직 공학 응용에 유망한 후보가 됨을 나타낸다.

목차 (Table of Contents)

• Table of Contents
• Abstract of Dissertation ii
• Table of Contents v
• List of Figures and Tables viii
• Chapter 1. Introduction to Graphene-based Nano Materials
• 1. 1. Graphene and Its Derivative 2
• 1. 2. Synthesis Methods 3
• 1. 2. 1. CVD Graphene 4
• 1. 2. 2. GQD synthesis methods 6
• 1. 3. Practical Applications of Graphene-based Nano materials 9
• 1. 3. 1. Application: GQDs in Fibrosis Treatment 9
• 1. 3. 2. Application: GQDs in MRI Contrast Agents 12
• 1. 3. 3. Application: Graphene in Tissue Engineering 16
• 1. 4. Paramagnetism of GQDs with EPR analysis 19
• 1. 5. References 22
• Chapter 2. Graphene quantum dots attenuate renal fibrosis through alleviating oxidative stress and restoring mitochondrial membrane potential
• 2. 1. Abstract 28
• 2. 2. Introduction 30
• 2. 3. Result 32
• 2. 4. Discussion 48
• 2. 5. Experimental 52
• 2. 6. Supplementary Information 64
• 2. 7. References 65
• Chapter 3. Dual Isotope-Labelled Graphene Quantum Dots for MRI and PET Imaging
• 3. 1. Abstract 71
• 3. 2. Introduction 72
• 3. 3. Result and Discussion 74
• 3. 4. Conclusion 92
• 3. 5. Experimental 92
• 3. 6. Supplementary Information 99
• 3. 7. References 104
• Chapter 4. Improved Osteogenesis of Human Adipose-Derived Stromal Cells on Hydroxyapatite-Mineralized Graphene Film
• 4. 1. Abstract 109
• 4. 2. Introduction 111
• 4. 3. Material and Methods 112
• 4. 4. Results 121
• 4. 5. Discussion 137
• 4. 6. Conclusion 140
• 4. 7. Supplementary Information 141
• 4. 8. Reference 145
• Chapter 5. Conculsion
• Abstract (in Korean) 152