Immunomodulatory hydrogels for enhanced therapeutic synergy with drugs

국문 초록 (Abstract)

본 연구는 생체재료의 고유한 면역조절 특성을 이용하여 종양 미세환경을 재조정하고, 수술 후 잔존암의 재발을 억제할 수 있는 면역조절 하이드로젤 플랫폼을 개발하는 것을 목표로 하였다. 암 수술 후 재발은 잔존 종양세포뿐 아니라 면역억제적 미세환경에 의해 유발되며, 이를 효과적으로 제어하기 위해서는 면역활성화와 면역억제의 균형적 조절이 필요하다. 본 연구에서는 천연 유래 생체재료의 면역조
절 가능성을 규명하고, 이를 기반으로 한 국소 면역치료용 하이드로젤 시스템을 구축하였다.
우선 다양한 생체재료를 스크리닝하여 면역세포 반응에 대한 내재적 조절능을 비교하였다. 그 결과 일부 재료는 수지상세포 성숙을 촉진하여 항원제시 능력과 T세포 활성화를 유도한 반면, 다른 재료들은 M2형 대식세포 분화를 억제하여 면역억제 환경을 완화하였다. 이러한 상반된 면역축의 특성을 기반으로, 본 연구에서는 면역활성화용 및 면역억제 억제용 두 가지 하이드로젤 시스템을 각각 설계하였다.
면역활성화 하이드로젤은 글라이콜 키토산을 메타크릴화하여 제작되었으며, DNA/독소루비신 복합체를 탑재함으로써 면역원성 세포사멸을 유도하고 수지상세포의 공자극 분자 발현을 증가시켰다. 이 하이드로젤은 마우스 유방암 절제 모델에서 종양의 재발과 전이를 억제하였으며, 수지상세포 및 T세포 활성화를 통해 생존율을 유의하게 향상시켰다.
면역억제 억제형 하이드로젤은 히알루론산에 티라민과 레스베라톨을 결합하여 제작된 주사형 하이드로젤로, 효소적 가교를 통해 우수한 생체적합성과 점진적 분해 특성을 나타냈다. 이 시스템은 레스베라톨의 면역억제 완화 기능을 유지하며 독소루비신의 안정적 방출을 구현하였고, M2 대식세포 마커인 CD206과 Argingase 1의 발현을 유의하게 감소시켰다. 또한 생체 내에서는 초기 24시간 동안 면역억제성 세포의 침윤을 억제하고, 7일 후에는 조절 T세포 감소가 나타나 초기 면역억제 환경을 완화하는 방향으로 작용하였다.
종합적으로, 본 연구는 생체재료가 단순한 약물전달 매트릭스를 넘어 면역환경을 능동적으로 조절할 수 있는 치료 플랫폼으로 활용될 수 있음을 제시하였다. 특히 면역활성화와 면역억제 억제라는 두 축을 상보적으로 제어하는 하이드로젤 시스템을 통해, 수술 후 암 재발 방지 및 항암면역 증진을 동시에 달성할 수 있는 전략을 확립하였다. 본 연구는 향후 면역항암제 병용, 수술 후 보조요법, 염증성 질환 치료 등 다양한 임상 응용으로 확장될 수 있는 기반 기술로서 의의가 있다.

다국어 초록 (Multilingual Abstract)

Biomaterials have traditionally served as conventional carriers in drug delivery, but growing evidence shows that many natural polymers possess intrinsic immunomodulatory properties that can influence innate immune cells. Hydrogels, with their high-water content, ECM-like structure, and tunable degradability, provide an ideal platform for integrating these biomaterial driven cues into localized therapeutic systems. When constructed from immune interactive biomaterials, hydrogels can function not only as drug depots but also as active regulators of the tumor immune microenvironment (TME). The TME is dominated by strong immunosuppressive forces, including M2 like tumor associated macrophages, regulatory T cells, and inhibitory cytokines that hinder dendritic cell (DC) maturation and cytotoxic T cell activation. Physical barriers such as dense extracellular matrix and disorganized vasculature further restrict drug penetration, contributing to postoperative recurrence. Therefore, biomaterials capable of stimulating DC activity or suppressing M2 polarization represent a rational strategy for reprogramming the local immune landscape.
In Chapter 2, a systematic screening of natural biomaterials was conducted to evaluate their intrinsic ability to activate DCs or inhibit M2 macrophage polarization. Glycol chitosan emerged as a strong promoter of DC maturation, whereas hyaluronic acid and resveratrol each showed inhibitory effects on IL4 driven M2 polarization with distinct but complementary patterns. These findings formed the basis for engineering two hydrogel platforms targeting these immune axes in a cell type–specific manner.
In Chapter 3, a DC targeting hydrogel system was developed using methacrylated glycol chitosan as a scaffold incorporating DNA/doxorubicin complexes to induce immunogenic cell death and amplify DC activation. In a 4T1-Luc2 breast cancer resection model, this hydrogel reduced postoperative recurrence, enhanced DC and T cell responses, and improved survival outcomes.
In Chapter 4, an M2 targeting hydrogel system was engineered by conjugating tyramine and resveratrol to hyaluronic acid to create an injectable, enzymatically crosslinkable matrix. This platform preserved the M2 modulating activity of resveratrol while enabling sustained doxorubicin release. It decreased M2 markers (CD206, and Arginase-1) in vitro and reduced early immunosuppressive cell infiltration and later Treg accumulation in vivo, indicating progressive remodeling of the suppressive TME.
Overall, this research demonstrates that the intrinsic immunoregulatory properties of biomaterials can be harnessed to design DC targeting and M2 targeting hydrogels that reshape the TME through localized, biomaterial driven immune modulation. These dual axis strategies provide a foundation for biomaterial-based localized immunotherapy aimed at preventing postoperative cancer recurrence and enhancing chemotherapeutic efficacy.

목차 (Table of Contents)

• Chapter 1. Introduction 1
• 1.1 Immune-regulating biomaterials 1
• 1.2 Hydrogels as a biomaterial-based drug delivery platform 5
• 1.3 Tumor immune microenvironment as a target for immune regulation 7
• 1.4 Key immune cell targets for biomaterial driven immune regulation 11
• 1.5 Research Rationale and Objectives 13
• Chapter 2. Screening of functional biomaterials for immunomodulation 15
• 2.1 Introduction 15
• 2.1.1 The tumor microenvironment (TME) and immune regulation 15
• 2.1.2 DCs: potent activators of anti-tumor immunity 18
• 2.1.3 Immunosuppressive role of M2 macrophages in tumor progression 19
• 2.1.4 Candidate biomaterials for immunomodulation 21
• 2.1.5 Rationale for screening immunomodulatory biomaterials 27
• 2.2 Methods 29
• 2.2.1 Materials 29
• 2.2.2 Animals and cell lines 29
• 2.2.3 in vitro DC activation analysis 30
• 2.2.4 in vitro M2 macrophage suppression analysis 31
• 2.2.5 Statistical analysis 33
• 2.3 Results 34
• 2.3.1 DC Activation analysis 34
• 2.3.2 M2 suppression analysis 41
• 2.4 Discussion 45
• 2.5 Conclusion 48
• Chapter 3. Development of dendritic cell targeting hydrogel systems 49
• 3.1 Introduction 49
• 3.1.1 Postsurgical cancer recurrence and the immunosuppressive tumor microenvironment 49
• 3.1.2 Dendritic cell activation as a key strategy in immune reprogramming 51
• 3.1.3 Glycol chitosan as a functional immunomodulatory biomaterial 53
• 3.1.4 Development of methacrylated glycol chitosan (MGC) Hydrogel 55
• 3.1.5 Combination strategy using DNA and doxorubicin 57
• 3.1.6 Research objective and chapter overview 61
• 3.2 Methods 63
• 3.2.1 Materials 63
• 3.2.2 Animals and cell lines 64
• 3.2.3 Synthesis of MGC 65
• 3.2.4 Fabrication of MGC hydrogels 65
• 3.2.5 Nuclear magnetic resonance spectroscopy 65
• 3.2.6 Morphology and porous scaffold analysis 66
• 3.2.7 Swelling behaviors of the MGC hydrogel 66
• 3.2.8 Rheological properties of MGC hydrogels 67
• 3.2.9 BMDC activation 67
• 3.2.10 Cytotoxicity 68
• 3.2.11 DNA/DOX complex preparation 69
• 3.2.12 In vitro release study 69
• 3.2.13 In vivo release study 70
• 3.2.14 In vivo degradation study 70
• 3.2.15 In vitro ICD assay 71
• 3.2.16 In vivo tumor models and treatment 71
• 3.2.17 In vivo bioluminescence and imaging 72
• 3.2.18 Evaluation of activated immune cells in vivo 72
• 3.2.19 Statistical analysis 74
• 3.3 Results 75
• 3.3.1 Synthesis of MGC hydrogel 75
• 3.3.2 DC activation by GC, MGC, and MGC hydrogel 82
• 3.3.3 Biocompatibility of MGC and MGC hydrogels 85
• 3.3.4 Degradation property of MGC hydrogel 87
• 3.3.5 Release profile of MGC hydrogel 90
• 3.3.6 In vitro ICD Assay 94
• 3.3.7 Antitumor effect of drug loaded MGC Hydrogels 97
• 3.3.8 Immune response by drug loaded MGC hydrogels in vivo 102
• 3.4 Discussion 106
• 3.5 Conclusion 113
• Chapter 4. Development of M2 macrophage targeting hydrogel systems 115
• 4.1 Introduction 115
• 4.1.1 Immunosuppressive tumor microenvironment and the role of M2 macrophages 115
• 4.1.2 Immune reprogramming via suppression of M2 macrophages 117
• 4.1.3 Design of a hyaluronic acidresveratrol composite immunomodulatory biomaterial 118
• 4.1.4 Combined chemo-immunotherapy using doxorubicin loaded HTR hydrogel 120
• 4.1.5 Research objectives and chapter overview 123
• 4.2 Methods 124
• 4.2.1 Materials 124
• 4.2.2 Animals and cell lines 125
• 4.2.3 Synthesis of hyaluronic acidtyramineresveratrol (HTR) 126
• 4.2.4 Fabrication of HTR hydrogel 127
• 4.2.5 Nuclear magnetic resonance spectroscopy 127
• 4.2.6 Rheological properties of HTR hydrogel 128
• 4.2.7 Swelling behaviors of the HTR hydrogel 129
• 4.2.8 In vitro M2 macrophage suppression analysis 129
• 4.2.9 Cytotoxicity 131
• 4.2.10 In vitro release study 132
• 4.2.11 In vivo release study 133
• 4.2.12 In vitro degradation behavior of the HTR hydrogel 133
• 4.2.13 In vivo degradation study 134
• 4.2.14 4T1 luc2 cytotoxicity 134
• 4.2.15 In vivo tumor models and treatment 135
• 4.2.16 In vivo bioluminescence and imaging 135
• 4.2.17 Evaluation of immune cell populations in vivo 136
• 4.2.18 Statistical analysis 137
• 4.3 Results 138
• 4.3.1 Synthesis and characterization of HTR Hydrogel 138
• 4.3.2 In vitro suppression of M2 macrophage polarization 148
• 4.3.3 Biocompatibility evaluation of HTR pre-gel and hydrogel 151
• 4.3.4 Degradation behavior of HTR hydrogel 153
• 4.3.5 In vitro and in vivo release profiles of HTR hydrogel 156
• 4.3.6 Functional validation of DOX loaded HTR hydrogel 159
• 4.3.7 Evaluation of in vivo antitumor efficacy of DOX loaded HTR hydrogel 161
• 4.3.8 Immune response induced by DOX loaded HA-RES hydrogel in vivo 166
• 4.4 Discussion 170
• 4.5 Conclusion 176
• Chapter 5. Discussion 178
• Chapter 6. Grand conclusion 182
• Reference 186
• 논문국문요약 204