Engineering targeted and stimuli-responsive drug delivery systems to advance precision therapy for colitis and cancer

다국어 초록 (Multilingual Abstract)

Precision drug delivery systems that utilize disease-specific microenvironmental and molecular indications represent a transformative strategy for improving therapeutic efficacy in complex inflammatory diseases and cancers. Conventional therapies often have drawbacks of limited tissue specificity, off-target effects, therapeutic resistance, and inadequate drug accumulation at pathological sites. These challenges are especially relevant in inflammatory bowel diseases and solid tumors where spatial heterogenous pathology, dynamic immune microenvironment, and cellular plasticity limit the durability and specificity of standard treatments. So, there is an urgent need for biologically intelligent delivery platforms that can respond selectively to the pathological signals.
Within this framework, the central objective of this thesis was to develop modular precision drug delivery strategies by integrating stimuli-responsive materials and receptor-targeted biologics with disease relevant triggers. This work utilized the hallmark features of pathological environments like oxidative stress, pH gradients, enzymatic activity, and receptor overexpression to spatially, cellularly and mechanistically controlled drug release. By combining rational material design and antibody engineering with a detailed understanding of disease biology, this work advances a multi-layered approach to therapeutic specificity.
In case of inflammatory bowel diseases, high reactive oxygen species production, epithelial barrier dysfunction, and dysregulated immune response cause chronic inflammatory and relapsing conditions. Conventional anti-inflammatory therapies are often limited by poor localization and dose-limiting toxicity. Microenvironment-responsive oral delivery systems were engineered in this study to address these limitations and synchronize drug release with oxidative and pH-related signals in the gastrointestinal tract. These platforms enabled localized drug delivery, enhanced mucosal retention, and targeted immunomodulation, highlighting the use of endogenous biochemical signals to improve therapeutic precision.
Additionally, this thesis also addressed key challenges of precision oncology, like tumor heterogeneity, immune evasion, and therapy-resistance cancer stem-like cells. Antibody drug conjugates (ADCs) were developed as targeted modality by combining monoclonal antibody’s specificity with the potent cytotoxic payloads. Rational target selection, linker-payload chemistry, and controlled conjugation ensured efficient intracellular drug release and favorable pharmacokinetics. ADC platforms targeting tumor-associated receptors and immune checkpoint molecules achieved selective tumor accumulation and enhanced anti-tumor efficacy, thereby modulating anti-tumor, immune-suppressive, and stemness associated pathways.
Overall, this work demonstrates that aligning delivery system design with disease-specific microenvironment and molecular features enables precise and effective therapeutic modulation, providing a translational framework for advanced precision medicine.

국문 초록 (Abstract)

정밀 약물 전달 시스템은 질환 특이적 미세환경과 분자적 신호를 활용하여 복잡한 염증성 질환과 암에서 치료 효능을 향상시키는 변혁적 전략을 제시한다. 기존 치료법은 제한된 조직 특이성, 표적 이탈(off-target) 효과, 치료 저항성, 병변 부위에서의 불충분한 약물 축적 등의 단점을 종종 보인다. 이러한 문제는 공간적으로 이질적인 병리, 역동적인 면역 미세환경, 세포 가소성이 표준 치료의 지속성과 특이성을 제한하는 염증성 장질환과 고형암에서 특히 두드러진다. 따라서 병리적 신호에 선택적으로 반응할 수 있는 생물학적으로 지능적인 전달 플랫폼이 시급히 요구된다.
이러한 틀 안에서, 본 논문의 핵심 목표는 자극 반응성 소재와 수용체 표적 생물의약품을 질환 연관 트리거와 통합하여 모듈형 정밀 약물 전달 전략을 개발하는 것이었다. 본 연구는 산화 스트레스, pH 구배, 효소 활성, 수용체 과발현과 같은 병리적 환경의 대표적 특징을 활용하여 약물 방출을 공간적·세포적·기전적으로 제어하였다. 합리적 소재 설계와 항체 공학을 질병 생물학에 대한 정밀한 이해와 결합함으로써, 본 연구는 치료 특이성을 위한 다층적 접근법을 진전시켰다.
염증성 장질환의 경우, 높은 활성산소종(ROS) 생성, 상피 장벽 기능 장애, 조절 실패한 면역 반응이 만성 염증과 재발성 상태를 초래한다. 기존 항염증 치료는 낮은 병변 국소화와 용량 제한 독성으로 종종 제약을 받는다. 이를 해결하기 위해 본 연구에서는 위장관의 산화 및 pH 관련 신호와 동기화된 약물 방출을 구현하는 미세환경 반응성 경구 전달 시스템을 설계하였다. 이러한 플랫폼은 국소화된 약물 전달, 점막 잔류성 향상, 표적 면역조절을 가능하게 하였으며, 내인성 생화학 신호를 활용해 치료 정밀성을 개선하는 접근을 부각시켰다.
또한 본 논문은 종양 이질성, 면역 회피, 치료 저항성 암 줄기세포 유사 세포와 같은 정밀 종양학의 핵심 과제도 다루었다. 항체-약물 접합체(ADC)는 단일클론 항체의 특이성과 강력한 세포독성 페이로드를 결합한 표적 치료 모달리티로 개발되었다. 합리적 표적 선정, 링커-페이로드 화학, 제어된 접합은 효율적인 세포내 약물 방출과 우수한 약동학을 보장하였다. 종양 연관 수용체와 면역관문 분자를 표적화하는 ADC 플랫폼은 선택적 종양 축적과 향상된 항종양 효능을 달성하여, 항종양성·면역억제성·줄기성 관련 경로를 조절하였다.
종합적으로, 본 연구는 전달 시스템 설계를 질환 특이적 미세환경 및 분자적 특징과 정렬함으로써 정밀하고 효과적인 치료 조절을 가능케 하며, 고도화된 정밀의학을 위한 번역(Translational)적 프레임워크를 제시한다.

목차 (Table of Contents)

• Chapter 1. Precision drug delivery platforms 1
• 1.2 Rationale of precision drug delivery in diverse disease conditions 2
• 1.3 Prevalence and clinical burden of ulcerative colitis (UC) 3
• 1.4 Clinical challenges and therapeutic needs in cancer 4
• 1.5 Aim of the thesis 5
• Chapter 2. ROS-responsive thioketal nanoparticle delivering system for targeted ulcerative colitis therapy with potent HDAC6 inhibitor, tubastatin A 6
• 2.1 Introduction 6
• 2.2 Materials and methods 9
• 2.2.1 Synthesis and characterization of ROS-responsive thioketal polymer 9
• 2.2.2 Development and characterization of ROS-responsive thioketal nanoparticle 9
• 2.2.3 Stimuli-responsive release of tubastatin A from TKNP 10
• 2.2.4 Cell culture 11
• 2.2.5 Intracellular uptake and internalization study 11
• 2.2.6 Cell viability assay 12
• 2.2.7 Intracellular ROS assay 12
• 2.2.8 Macrophage activation study 13
• 2.2.9 Cytokine release assay 13
• 2.2.10 Animals 13
• 2.2.11 In vivo imaging and biodistribution study in colitis bearing mice 13
• 2.2.12 In vivo treatment studies in colitis induced mouse model 14
• 2.2.13 Myeloperoxidase activity assay 15
• 2.2.14 Histopathological evaluation 15
• 2.2.15 Quantification of cytokines in colonic tissue 16
• 2.2.16 Statical analysis 16
• 2.3 Results 17
• 2.3.1 Synthesis and characterization of thioketal polymer 17
• 2.3.2 Development and characterization of tubastatin A loaded thioketal nanoparticle 19
• 2.3.3 ROS-responsive release of TUBA from TUBA-TKNP 19
• 2.3.4 In vitro cellular uptake study 21
• 2.3.5 In vitro cell viability and intracellular ROS study 23
• 2.3.6 In vitro macrophage polarization assay 25
• 2.3.7 In vitro cytokines release assay 27
• 2.3.8 In vivo biodistribution and retention of TKNP in GI tract of colitis bearing mice 29
• 2.3.9 In vivo therapeutic efficacy of TUBA-TKNP in mice with DSS-induced colitis 32
• 2.3.10 MPO activity in colonic tissue 35
• 2.3.11 Histopathological evaluation 37
• 2.3.12 Cytokines level in colonic tissue 39
• 2.4 Discussion 41
• 2.5 Conclusion 47
• Chapter 3. Colon Specific Delivery of EGCG via Engineering pH-Responsive Chitosan Microparticles for Ulcerative Colitis Therapy 48
• 3.1 Introduction 48
• 3.2 Materials and Methods 51
• 3.2.1 Materials 51
• 3.2.2 Synthesis of Eudragit®S100 coated EGCG loaded microparticle 51
• 3.2.3 Physicochemical characterization of microparticles 52
• 3.2.4 Determination of LC% and EE% of EGCG in Chitosan MPs 53
• 3.2.5 In vitro release of EGCG from MPs 54
• 3.2.6 Cells culture 54
• 3.2.7 Biocompatibility study 54
• 3.2.8 Cellular uptake analysis 55
• 3.2.9 In vitro macrophage polarization assay 56
• 3.2.10 Intracellular ROS generation assay 56
• 3.2.11 Wound healing scratch assay 57
• 3.2.12 Animal studies 58
• 3.2.13 Establishment of the DSS-induced colitis mouse model 58
• 3.2.14 Biodistribution study 59
• 3.2.15 In vivo therapeutic efficacy study in colitis bearing mice 59
• 3.2.16 Statistical analysis 60
• 3.3 Results 61
• 3.3.2 Preparation and characterization of EG@EuCS 61
• 3.3.3 pH responsive release of the EGCG from EG@EuCS MPs 65
• 3.3.4 Biocompatibility assessment of EGCG formulations 68
• 3.3.5 Enhanced cellular uptake and lysosomal trafficking of chitosan microparticles 70
• 3.3.6 Effects of EGCG-loaded chitosan microparticles on macrophage polarization 72
• 3.3.7 EG@CS MPs effectively suppressed LPS-induced oxidative stress in macrophages 75
• 3.3.8 Chitosan based EGCG delivery stimulates epithelial wound closure in oxidative stress induced injury 77
• 3.3.9 In vivo biodistribution and GI retention of Eudragit coated Cy5.5-loaded chitosan microparticle 79
• 3.3.10 EG@EuCS exerted significant therapeutic efficacy against UC in vivo 82
• 3.4 Discussion 86
• 3.5 Conclusion 90
• Chapter 4. CD44-Directed Antibody Drug Conjugates Reprogram Tumor Transcriptomes to Suppress Cancer Stem Cell-Driven Ovarian Cancer Progression 91
• 4.1 Introduction 91
• 4.2 Materials and Methods 94
• 4.2.1 Cell Culture 94
• 4.2.2 Synthesis of DOX-Valine Citrulline Drug-Linker Species 94
• 4.2.3 Development and Characterization of Antibody-Drug Conjugates 94
• 4.2.4 Analysis of Cell Surface CD44 Expression Levels in Ovarian Cancer Cells 95
• 4.2.5 In vitro Cell Antigen Binding Assay 96
• 4.2.6 Cellular Uptake, Internalization, and Lysosomal Trafficking 97
• 4.2.7 In vitro Cytotoxicity and Live/Dead Assay 98
• 4.2.8 In vitro Apoptosis Evaluation 98
• 4.2.9 Cell Cycle Arrest Analysis 99
• 4.2.10 Establishment of the Xenograft Tumor Model 99
• 4.2.11 Biodistribution Study 100
• 4.2.12 In vivo Evaluation of Anti-Tumor Effects 101
• 4.2.13 Histopathological and Immunohistochemical Analyses 101
• 4.2.14 Transcriptomic Profiling and mRNA Sequencing Analysis 102
• 4.2.15 Statistical analysis 103
• 4.3 Results 104
• 4.3.1 Synthesis and Characterization of the MC-VC-PAB-DOX Payload 104
• 4.3.2 Development and Characterization of the CD44-VC-DOX Antibody-Drug Conjugate 107
• 4.3.3 Differential CD44 Expression in Ovarian Cancer Cell Lines 111
• 4.3.4 The CD44-VC-DOX ADC Binds Specifically to the CD44 Cell Surface Antigen 113
• 4.3.5 CD44-Mediated Cellular Uptake and Intracellular Trafficking of CD44-VC-DOX 115
• 4.3.6 Assessment of the Cytotoxicity and Therapeutic Efficacy of CD44-VC-DOX in CD44-Positive and -Negative Ovarian Cancer Cells 118
• 4.3.7 CD44-VC-DOX-Induced Apoptosis and Cell Cycle Arrest in CD44 Overexpressing Ovarian Cancer Cells 120
• 4.3.8 In vivo and Ex vivo Biodistribution of CD44-VC-DOX 123
• 4.3.9 CD44-VC-DOX Inhibited Tumor Growth in SKOV3ip1 Tumor-Bearing Xenograft Mice 126
• 4.3.10 In vivo Proliferation, DNA Fragmentation, and Histopathological Evaluation following the CD44-VC-DOX Treatment 129
• 4.3.11 Molecular Characterization of Tumors via RNA-seq following CD44-VC-DOX Treatment 131
• 4.4 Discussion 138
• 4.5 Conclusion 143
• Chapter 5. A Site-Specific PD-L1–ADC Combining Potent Cytotoxicity with Inhibition of Immune Evasion 144
• 5.1 Introduction 144
• 5.2. Materials and methods 147
• 5.2.1 Materials 147
• 5.2.2 Synthesis of drug-linker payload 147
• 5.2.3 Development of Antibody Drug Conjugates 148
• 5.2.4 Cell culture 148
• 5.2.5 Cell surface PD-L1 expression analysis 149
• 5.2.6 In vitro cell surface antigen binding analysis 150
• 5.2.7 Cellular Uptake, Internalization, and Lysosomal Trafficking 150
• 5.2.8. In vitro cell cytotoxicity and live/dead assay 151
• 5.2.9 In vitro cell apoptosis evaluation 152
• 5.2.10 Cell cycle arrest analysis 153
• 5.2.11 Construction and characterization of 3D TNBC spheroid models 153
• 5.2.12 Cell viability of 3D tumor spheroid following treatment 154
• 5.2.13 Animal experiment and ethical permissions 155
• 5.2.14 CD8+ T cell-mediated cytotoxic effects on cancer cells in vitro 155
• 5.2.15 In vivo biodistribution study and tumor targeting evaluation 156
• 5.3 Results 158
• 5.3.1 Characterization of Glu-MMAE drug linker payload moiety 158
• 5.3.2 Characterization of site-specifically conjugated PD-L1-Glu-MMAE ADC 160
• 5.3.3 High surface expression of PD-L1 on TNBC cell lines 163
• 5.3.4 Cellular binding, uptake and receptor-mediated internalization of PD-L1 targeted ADC 165
• 5.3.5 In vitro anti-tumor efficacy of PD-L1 ADC 168
• 5.3.6 PD-L1 ADC induced apoptosis by enhancing cell cycle arrest at G2-M phase in MDA-MB-231 cells 170
• 5.3.7 Development and optimization of 3D tumor spheroid model 4T1 TNBC cells 173
• 5.3.8 Live/dead assessment confirms enhanced cytotoxicity of PD-L1 ADC on 3D tumor spheroid models 175
• 5.3.9 PD-L1 ADC enhances CD8+ T-cell mediated tumor killing through PD-1/PD-L1 axis blockade 177
• 5.3.10 PD-L1 ADC showed preferential tumor accumulation in vivo in 4T1 tumor bearing mice 181
• 5.4. Discussion 184
• 5.5 Conclusion 187
• Chapter 6. Thesis Summary and Future Perspectives 188
• 6.1 General Conclusion 188
• 6.2 Conceptual and Design Implications 191
• 6.3 Limitations and future perspectives 193
• References 195
• 논문요약 228