A High-Resolution Apicobasal Imaging Platform for Visualizing Intracellular Trafficking of Brain-Targeted Therapeutics Across the Blood?Brain Barrier

다국어 초록 (Multilingual Abstract)

Receptor-mediated transcytosis (RMT) represents a promising strategy for delivering macromolecular and colloidal therapeutics across the blood–brain barrier (BBB). However, mechanistic elucidation of RMT remains limited by the difficulty of visualizing subcellular trafficking pathways. Conventional imaging approaches either lack sufficient spatial resolution or require costly, technically complex instrumentation. Here, we report a cell swelling and upright mounting (CSUM-based) imaging approach that reorients the Z-axis into the high-resolution XY-plane using standard confocal microscopy, enabling direct RMT visualization without computational reconstruction or specialized hardware. To assess the morphological stability of cells during the swelling step, we conducted live-cell confocal imaging using CellMask and MitoTracker across different osmolarity environments. These measurements demonstrated uniform membrane expansion and preserved mitochondrial distribution under the selected hypotonic condition, without obvious disruption of subcellular organization. Additionally, F-actin staining showed that overall cytoskeletal architecture remained intact, indicating that the CSUM workflow maintains near-native cellular morphology during imaging within the tested time. Using this validated approach, we tracked intracellular trafficking of transferrin (Tf) and anti-transferrin receptor antibody (TfR Ab) as model cargos using our CSUM- based imaging approach via compartment-specific markers and time-resolved co- localization analysis. This approach resolved cargo-containing vesicles traversing from the apical to basolateral membranes. The CSUM approach provides a simple yet effective platform for high-resolution visualization of membrane transport and vesicle dynamics, offering broad applicability to drug delivery research and the design of brain-targeted therapeutics. Keywords: Blood-brain barrier, receptor-mediated transcytosis, intracellular trafficking, confocal microscopy, vesicle transport, direct cellular visualization, brain drug delivery system

국문 초록 (Abstract)

Receptor-mediated transcytosis (RMT)는 거대분자 및 나노입자 기반 약물을 뇌혈관장벽 (blood–brain barrier, BBB)을 통해 전달하기 위한 유망한 전략으로 여겨진다. 그러나 RMT의 세부 기전을 규명하기 위해서는 세포내 소기관을 가로지르는 이동 경로를 정밀하게 관찰해야 하며, 기존 영상 기법은 공간 해상도의 한계 또는 고가·고난도의 장비 의존성으로 인해 이러한 분석에 제약을 받아왔다. 본 연구에서는 세포 팽윤과 수직 배치 기법을 결합한 CSUM (cell swelling and upright mounting) 기반 이미징 기법을 제안한다. 이 방법은 일반적인 공초점 현미경을 활용하여 세포의 Z축을 고해상도의 XY 평면으로 재배치함으로써, 별도의 계산적 재구성이나 특수 장비 없이도 RMT 과정을 직접적으로 시각화할 수 있게 한다. 또한 세포 팽윤 과정이 세포 구조에 미치는 영향을 평가하기 위해, 다양한 삼투 조건에서 CellMask와 MitoTracker를 이용한 Live-cell 이미징을 수행한 결과 세포막 확장과 미토콘드리아 분포가 안정적으로 유지됨을 확인하였다. 추가적인 F-actin 염색에서도 세포골격 구조의 보존이 확인되어, CSUM 과정이 세포의 형태적 무결성을 유지한 상태에서 영상화를 가능하게 함을 검증하였다. 이 최적화된 CSUM 기반 기법을 활용하여, transferrin (Tf)과 anti-transferrin receptor antibody (TfR Ab)의 세포내 이동을 구획 특이적 마커 및 시간 의존적 공존 분석을 통해 추적하였다. 이를 통해 물질을 함유한 소포가 세포의 apical 면에서 basolateral 면으로 이동하는 경로를 직접 확인할 수 있었다. 종합적으로, CSUM 기법은 막 수송 및 소포 동역학을 고해상도로 관찰할 수 있는 간단하면서도 강력한 영상 플랫폼을 제공하며, 뇌 표적 약물전달 연구 및 BBB 통과 기전 규명에 폭넓게 활용될 수 있는 잠재력을 지닌다. 주요어: 뇌혈관장벽; 수용체 매개 세포횡단수송; 세포내 수송; 공초점 현미경; 세포내 시각화; 뇌 약물전달시스템

목차 (Table of Contents)

• ABSTRACT v
• LIST OF TABLES x
• LIST OF FIGURES xi
• LIST OF ABBREVIATIONS xiii
• 1. INTRODUCTION 15
• 1.1. Blood-Brain Barrier 15
• 1.1.1. BBB structure and function 15
• 1.1.2. Drug delivery challenges across the BBB 16
• 1.2. Transport pathways across the BBB 19
• 1.2.1. Paracellular pathway 19
• 1.2.2. Carrier-mediated transport 19
• 1.2.3. Adsorptive-mediated transcytosis 20
• 1.2.4. Receptor-mediated transcytosis 20
• 1.2.4.1. Transferrin receptor-mediated transcytosis 21
• 1.2.4.2. Comparative approaches using Tf and TfR Ab in brain drug delivery 23
• 1.3. Intracellular trafficking mechanisms 26
• 1.3.1. Overview of endosomal pathways 26
• 1.3.2. Rab GTPases 26
• 1.3.3. SNARE proteins 27
• 1.4. Comparative analysis of cellular imaging techniques 28
• 1.4.1. Current cellular imaging techniques and limitations 28
• 1.4.2. Imaging technique applications in BBB research 32
• 1.5. Aims of the study 35
• 2. MATERIALS AND METHODS 38
• 2.1. Materials 38
• 2.2. Methods 40
• 2.2.1. In vitro BBB permeability of Tf and TfR Ab using BBB Transwell model 40
• 2.2.2. Enzyme-linked immunosorbent assay (ELISA) 41
• 2.2.3. Optimization of cell swelling conditions using a hypotonic solution 42
• 2.2.3.1. Bright-field time-course imaging of cell morphology 43
• 2.2.3.2. Live-cell imaging of membrane and mitochondrial changes 43
• 2.2.3.3. Confocal imaging for measurement of cell height 43
• 2.2.3.4. Confocal imaging for F-actin cytoskeleton 44
• 2.2.4. Compartment-specific immunocytochemistry for tracking intracellular cargo trafficking 45
• 2.2.5. Upright mounting process and laser scanning confocal microscopy (LSCM) imaging analysis 45
• 2.2.6. Statistical analysis 46
• 3. RESULTS 47
• 3.1. In vitro BBB permeability of Tf and TfR Ab 47
• 3.2. Morphological and structural responses of bEnd.3 cells to hypotonic conditions 51
• 3.2.1. Bright-field and DIC imaging reveal early-stage morphological changes under hypotonic conditions 51
• 3.2.2. Live-cell imaging of membrane and mitochondrial responses under hypotonic conditions 54
• 3.2.3. Evaluation of F-actin integrity under hypotonic conditions 56
• 3.2.4. Quantitative analysis confirms hypotonic-induced increase in cell height 58
• 3.3. CSUM-based imaging enables direct visualization of transcytotic transport 61
• 3.4. Distinct temporal trafficking patterns of Tf and TfR Ab through endosomal compartments 63
• 3.4.1. Early endosome association patterns differ between Tf and TfR Ab 63
• 3.4.2. Late endosome/Lysosome trafficking profiles diverge between Tf and TfR Ab 67
• 3.4.3. Recycling endosome routing patterns differ between Tf and TfR Ab 70
• 3.4.4. Basolateral fusion dynamics reveal differences in transcytosis completion between Tf and TfR Ab 73
• 4. DISCUSSION 82
• 5. CONCLUSION 88
• 6. BIBLIOGRAPHY 90
• ABSTRACT IN KOREAN 102