Antibody fragment-conjugated nanoparticles for enhanced cellular targeting and precision drug delivery

다국어 초록 (Multilingual Abstract)

This dissertation presents the development of a biomaterial-based nanoparticle system designed to integrate precise targeting with controllable drug release. To achieve precise targeting, F(ab’)₂ antibody fragments were conjugated to biodegradable and biocompatible poly(lactic-co-glycolic acid) (PLGA) nanoparticles. This design allows independent control of two functional dimensions: the surface layer, which determines ligand orientation, density, and binding specificity; and the polymeric core, which controls drug encapsulation level, composition, and release kinetics. Through this dual-level modulation, the system enables alignment between biological targeting and pharmacodynamic profiles.
In Chapter 2, a controlled head-to-head comparison between F(ab’)₂-conjugated nanoparticles and whole-IgG–conjugated nanoparticles was
performed under strictly matched physicochemical conditions, including size and zeta potential. Following intravenous administration in mice, F(ab’)₂-conjugated nanoparticles exhibited superior in vivo targeting efficiency, with markedly increased accumulation in circulating and splenic target immune cells. These findings establish the fragment-based conjugation strategy as a more effective targeting approach than whole-antibody–based formulations.
In Chapter 3, the platform was applied to an inflammatory lung disease model to evaluate therapeutic utility. A sequential treatment strategy targeting neutrophil extracellular traps (NETs) and neutrophil elastase (NE) was developed using a combination of an inhalable DNase I–polydopamine nanoparticle to reduce NET formation, followed by intravenous delivery of sivelestat-loaded anti-Ly6G F(ab’)₂ nanoparticles to suppress neutrophil hyperactivation. This sequential approach decreased neutrophil burden, lowered NET/NE-associated biomarkers, improved lung function, and reduced fibrotic remodeling as indicated by hydroxyproline levels, while maintaining a favorable safety profile.
In chapter 4, the system was extended to oncology using anti-PD-L1 F(ab’)₂-modified nanoparticles loaded with doxorubicin. These nanoparticles achieved enhanced intratumoral accumulation, improved cytotoxicity, and superior tumor growth inhibition at matched drug doses. Immune profiling showed increased effector T cell activation and decreased exhaustion markers, indicating beneficial reprogramming of the tumor immune microenvironment. Additionally, systemic toxicity markers in cardiac and splenic tissues were reduced, demonstrating an improved therapeutic window.
Collectively, this study developed modular nanoplatform that maintains precise antigen recognition through F(ab’)₂ ligands while independently controlling drug release behavior. By decoupling targeting from release, the system allows disease-specific optimization of therapeutic performance. The platform demonstrated applicability in both immune and tumor models, simultaneously improving selectivity, efficacy, and safety. Furthermore, it extends the core design principles of antibody therapeutics into a clinically validated nanoparticle system, providing a conceptual framework that bridges antibody based therapy with biomaterials based drug delivery technology.

목차 (Table of Contents)

• Chapter 1. Introduction 1
• 1.1 Motivation 1
• 1.2 Nanoparticle based drug delivery 3
• 1.2.1 Biodegradable polymeric nanoparticles and PLGA 3
• 1.2.2 Surface chemistry and modular functionalization 6
• 1.3 Antibody-based precision therapeutics 8
• 1.3.1 Antibody drug conjugates 10
• 1.3.2 Antibody fragments as complementary targeting ligands 14
• 1.4 Scope and aim 18
• Chapter 2. Comparison of F(ab')₂ and Whole IgG-Conjugated Nanoparticles for Targeted Delivery Efficiency 22
• 2.1 Introduction 22
• 2.2 Methods 26
• 2.2.1 Preparation and characterizations of the conjugated nanoparticles 26
• 2.2.2 Conjugation of the antibodies to the nanoparticles 27
• 2.2.3 In vitro binding stability of antibodynanoparticles conjugates 28
• 2.2.4 Animals 29
• 2.2.5 Evaluation of the immune cell-targeting ability of the nanoparticles in vitro 30
• 2.2.6 Evaluation of the immune cell-targeting efficiency of the nanoparticles in vivo 30
• 2.2.7 Statistical Analyses 31
• 2.3 Results 32
• 2.3.1 Preparation and characterization of the PLGA-PEG-COOH nanoparticle and PLGA-PEG-Mal nanoparticle 32
• 2.3.2 Measurement of the number of antibodies conjugated to the surfaces of the NPs 40
• 2.3.3 In vitro binding stability of the antibodies conjugated to the nanoparticles 45
• 2.3.4 Immune cell targeting efficiency of the nanoparticles in vitro 48
• 2.3.5 Immune cells targeting efficiency of the nanoparticles in vivo 55
• 2.4 Conclusion 59
• Chapter 3. Neutrophil Targeting PLGA Nanoparticle for Pulmonary Fibrosis Treatment 60
• 3.1 Introduction 60
• 3.2 Methods 66
• 3.2.1 Preparation and characterization of DNase-I@PDA NP 66
• 3.2.2 Preparation of Neutrophil targeted Siv@PLGA NP 67
• 3.2.3 Characterization of Neutrophil targeted Siv@PLGA NP 68
• 3.2.4 Animals and husbandry 70
• 3.2.5 Aerosolized LPS-induced pulmonary fibrosis mouse model 70
• 3.2.6 Quantification of DNase-I using Enzyme-Linked Immunosorbent Assay (ELISA) in murine plasma and bronchoalveolar lavage fluid (BALF) 71
• 3.2.7 Quantification of plasma cfDNA 71
• 3.2.8 In vivo biodistribution 72
• 3.2.9 Cytokine array 72
• 3.2.10 Hematoxylin & eosin staining 73
• 3.2.11 Orange G staining 74
• 3.2.12 Trichrome staining 74
• 3.2.13 Quantitative Real-Time PCR Analysis 75
• 3.2.14 Hydroxyproline assay 76
• 3.2.15 Immunohistochemistry 76
• 3.2.16 Lung function assessment 77
• 3.2.17 Body weight and survival rate 78
• 3.2.18 Sample collection and neutrophil isolation of COVID-19 patients' plasma 79
• 3.2.19 NET ELISA 79
• 3.2.20 MPO ELISA 80
• 3.2.21 ELISA for DNase-I activity 80
• 3.2.22 Statistical analysis and software 81
• 3.3 Results 82
• 3.3.1 NPs preparation and characterization 82
• 3.3.2 Amelioration of acute immune responses in LPS-induced lung injury through early administration of DNase-I 94
• 3.3.3 Pulmonary retention and coadministration of DNase-I@PDA NP and Siv@PLGA NP 102
• 3.3.4 Relieved pathological phenotypes of pulmonary fibrosis in vivo 115
• 3.3.5 Therapeutic efficacy of NPs on neutrophils of COVID 19 patients 124
• 3.4 Conclusion 127
• Chapter 4. Tumor Cell Targeting PLGA Nanoparticle for Anti-Cancer Chemotherapy 128
• 4.1 Introduction 128
• 4.2 Methods 134
• 4.2.1 Preparation and characterization of PD-L1+Dox@NP 134
• 4.2.2 Release profile and encapsulation efficiency of Dox@NP 135
• 4.2.3 In vitro tumor cell targeting efficiency 137
• 4.2.4 In vivo assessment of NP accumulation in tumor tissue 138
• 4.2.5 In vitro and in vivo anti-cancer efficiency 138
• 4.2.6 Evaluation of in vivo T cell activation 139
• 4.2.7 Quantification of oxidative stress in heart and kidney tissues 141
• 4.2.8 Animals 141
• 4.2.9 Statistical Analyses 142
• 4.3 Results 143
• 4.3.1 Characterization of Dox@NP 143
• 4.3.2 Tumor targeting efficiency of PD-L1+Dox@NP 148
• 4.3.3 Anti-cancer effect of PD-L1+Dox@NP 153
• 4.3.4 Enhanced T cell activation and reduced immunosuppression by PD-L1+Dox@NP 157
• 4.3.5 Evaluation of systemic biosafety and oxidative stress markers after PD-L1+Dox@NP treatment 167
• 4.4 Conclusion 170
• Chapter 5. Conclusion 172
• Reference 176
• 논문요약 186