Nanomaterial-Functionalized Impedance Biosensors for Continuous 3D Tissue Monitoring and the Assessment of Immunomodulatory Therapeutic Nanocomposites

다국어 초록 (Multilingual Abstract)

Conventional two-dimensional (2D) cell cultures and endpoint assays fail to capture the kinetic dynamics required to accurately evaluate cellular microenvironments and therapeutic efficacy. This thesis addresses these limitations by establishing a unified theragnostic framework built upon two core pillars: the development of continuous electrical impedance biosensors for label-free monitoring, and the engineering of therapeutic nanocomposites for enhanced intracellular delivery. Centered on the macrophage immune axis, the experimental work systematically progresses from 2D biosensing toward the real-time evaluation of advanced nanotherapeutics in three-dimensional (3D) tumor and epidermal tissue models. The foundational monitoring capabilities were established using indium tin oxide (ITO) microelectrodes. First, continuous impedance monitoring successfully resolved M0, M1, and M2 macrophage polarization dynamics in real-time without destructive sampling. This platform was then progressively enhanced via a dual nanomaterial coating (Siglec-1/β- cyclodextrin/reduced graphene oxide) to continuously detect Neu5Ac xeno- antigen expression on genetically engineered red blood cells, demonstrating high impedimetric sensitivity. Transitioning to 3D models, the thesis applied continuous impedance monitoring to evaluate the efficacy of newly developed nanocomposites designed to overcome the delivery barriers of complex tissues. For the 3D tumor model, an electropolymerized polydopamine (PDA) coating and PDMS microwell array were applied to the ITO platform, ensuring stable spheroid adhesion. By tuning the applied frequency to 10 kHz to penetrate the 3D mass, the platform continuously tracked the enhanced penetration and superior anticancer kinetics of cryptotanshinone-graphene quantum dot (CPT-GQD) nanocomposites compared to free drugs. Applying this identical strategy to a 3D epidermal tissue model, a custom fixed-geometry transepithelial/transendothelial electrical resistance (TEER) device was developed to overcome the geometric variability of commercial electrodes, to continuously monitor tight junction integrity. This continuous TEER monitoring was used to evaluate a second advanced delivery system: R9 cell-penetrating peptide-engineered exosomes. Just as the GQDs enhanced CPT delivery in tumors, molecular dynamics simulations and continuous 18-day impedance monitoring confirmed that R9 surface- functionalization significantly enhanced the cellular uptake and delivery of wound-healing miRNAs from M2 macrophages and mesenchymal stem cells, promoting superior skin barrier regeneration. Collectively, this thesis demonstrates that integrating continuous impedance monitoring with 3D tissue models is a critical necessity for the accurate, real-time preclinical evaluation of nanomaterial-enhanced drug and exosome delivery systems.

목차 (Table of Contents)

• 1 Introduction 35
• 1.1 Three-Dimensional Cell Culture Models: From Conventional Limitations Toward Physiologically Relevant Platforms 35
• 1.2 Immunomodulation and Immunotherapy: Biological Rationale and Therapeutic Strategies 39
• 1.3 Immunomodulatory Therapeutic Nanocomposites: Design Rationale and Biological Evaluation Framework 43
• 1.3.1 Nanomaterial–Drug Nanocomposites for Anticancer Delivery: GQD– Cryptotanshinone 44
• 1.3.2 Vesicle–Peptide Nanocomposites for Skin Regeneration and Protection: R9- Conjugated Exosomes 46
• 1.4 Advances in Electrical Biosensors for Biological Monitoring, Drug Screening, and Therapy Evaluation 48
• 1.5 Electrical Impedance Biosensors for Continuous Cell Monitoring: Principles, Historical Development, and Selection Rationale 51
• 1.6 Fabrication of ITO Electrode Platforms, Nanomaterial Coating Strategies, and TEER Device Integration 54
• 1.7 Computational Validation of Therapeutic Nanocomposite Design: Molecular Docking and Molecular Dynamics Simulations 56
• 1.8 Thesis Outline, Research Aims, and Original Contributions 58
• 2 MACROPHAGE POLARIZATION OF THP-1 MONOCYTES ON AN INDIUM TIN OXIDE ELECTRODE: LABEL-FREE AND REAL-TIME ELECTRICAL IMPEDANCE MONITORING 62
• 2.1 Introduction 62
• 2.2 Experiment 67
• 2.2.1 Reagents 67
• 2.2.2 Cell culture 67
• 2.2.3 Flow cytometry 68
• 2.2.4 Immunofluorescence 68
• 2.2.5 Cell Impedance Measurement 69
• 2.2.6 Statistical analysis 69
• 2.3 Results and Discussion 70
• 2.3.1 Dilution fraction 70
• 2.3.2 Monitoring THP-1 macrophage differentiation 73
• 2.3.3 Characterization of various kinds of THP-1-originated macrophages using flow cytometry and fluorescence microscopy 75
• 2.4 Conclusions 78
• 2.5 Supporting information 79
• 3 A Novel Siglec-1/β-CD/rGO Impedimetric Biosensor to Identify Neu5Ac Expression in Red Blood Cells 82
• 3.1 Introduction 82
• 3.2 Materials and Methods 85
• 3.2.1 Reagents 85
• 3.2.2 RBC preparation 86
• 3.2.3 Sialic acid quantification using multiple reaction monitoring-mass spectrometry (MRM-MS) 86
• 3.2.4 Fabrication platform 86
• 3.2.5 Real-time impedimetric monitoring 87
• 3.2.6 Scanning electron microscopy (SEM) 87
• 3.2.7 The enzyme-linked immunosorbent assay (ELISA) 87
• 3.2.8 Statistical Analysis 88
• 3.3 Results and Discussions 88
• 3.3.1 Characterization of Siglec-1/β-CD/rGO Deposition on ITO Microelectrodes 88
• 3.3.2 Neu5Ac and Neu5Gc levels in red blood cells 91
• 3.3.3 Real-Time Impedimetric Detection with β-CD/rGO/ITO Platform 92
• 3.3.4 Real-Time Neu5Ac Detection on Different RBC Types 96
• 3.3.5 Reusability and Specificity 98
• 3.3.6 Implications and Applications 100
• 3.4 Conclusion 101
• 3.5 Support Information 101
• 4 Real-time Impedimetric Monitoring of Three Dimensional Cancer Spheroids Utilizing a PDA-Functionalized Microelectrode Platform Integrated with Graphene Quantum Dot-Based Nanocomposites 108
• 4.1 Introduction 108
• 4.2 Materials & Methods 114
• 4.2.1 Materials 114
• 4.2.2 PDMS microwell with ITO manufacturing 114
• 4.2.3 Electro-polymerization procedure and electrode preparation 115
• 4.2.4 Visualization showing the interaction between CPT-GQDs and cancer receptors 116
• 4.2.5 Preparation and functionalization of CPT-GQD conjugates 116
• 4.2.6 Physicochemical characterisation methods 116
• 4.2.7 Cell culture 117
• 4.2.8 Gene Expression Assays 117
• 4.2.9 MCF-7 cell viability: Flow cytometry, MTT, and ATP luminescence 118
• 4.2.10 Fluorescent staining 118
• 4.2.11 Real-time impedimetric methods for monitoring the MCF-7 119
• 4.2.12 Statistical analysis 120
• 4.3 Results and Discussion 120
• 4.3.1 PDA deposition characterization on an ITO microelectrode 120
• 4.3.2 CPT-GQDs Development and Characterization 121
• 4.3.3 In silico prediction of anticancer drug interactions with MCF-7 receptors 124
• 4.3.4 Gene Expression Analysis in MCF-7 Breast Cancer 127
• 4.3.5 Effects on MCF-7 viability using various assessment techniques 128
• 4.3.6 Real-time impedimetric monitoring of MCF-7 proliferation 129
• 4.3.7 CPT and CPT-GQD uptake and inhibitory effects are monitored in real time using impedimetry 132
• 4.4 Conclusions 136
• 4.5 Supporting Information 138
• 5 Peptide-Engineered Exosomes Enable Quantitative Regeneration Analysis in 3D Epidermal Tissue via Integrated TEER Monitoring 143
• 5.1 Introduction 143
• 5.2 Materials & Methods 146
• 5.2.1 Cell culture and 3D epidermal model establishment 146
• 5.2.2 Exosome isolation and R9 peptide surface engineering 147
• 5.2.3 Molecular dynamics simulations 148
• 5.2.4 TEER device design and fabrication 151
• 5.2.5 TEER monitoring 152
• 5.2.6 Cell proliferation and viability assessment 152
• 5.2.7 Small RNA sequencing and bioinformatic analysis 153
• 5.2.8 RNA extraction and quantitative real-time PCR 154
• 5.2.9 Western blot analysis 155
• 5.2.10 TEM 155
• 5.2.11 Physicochemical characterization 156
• 5.2.12 Statistical analysis 157
• 5.3 Result 157
• 5.3.1 Small RNA sequencing reveals distinct miRNA cargo in MSC and M2 mac rophage exosomes 157
• 5.3.2 Molecular dynamics simulation of R9 peptide–POPC membrane interaction 161
• 5.3.3 EDC/NHS covalent conjugation produces stable R9–tetraspanin linkages with scaffold-dependent conformational control 162
• 5.3.4 Isolation, engineering, and physicochemical characterization of exosomes 166
• 5.3.5 R9 surface engineering enhances exosome-driven keratinocyte proliferation and wound healing in vitro 170
• 5.3.6 R9 surface engineering enhances exosome-driven keratinocyte proliferation and barrier formation in a 3D epidermal model 176
• 5.3.7 R9-functionalized exosomes protect and regenerate 3D epidermal tissue against oxidative damage 181
• 5.4 Discussion 186
• 5.5 Supporting Information 191
• 6 Conclusions and Future Perspectives 200
• 6.1 Overall Conclusions 200
• 6.2 Limitations and Critical Evaluation 205
• 6.3 Future Perspectives 207
• References 212
• Acknowledgments 244
• Curriculum Vitae 246