Therapeutic evaluation of adipose tissue-derived nano lipid droplets in inflammatory bowel disease

다국어 초록 (Multilingual Abstract)

Inflammatory bowel disease (IBD) is a chronic intestinal disorder characterized by oxidative stress, epithelial barrier dysfunction, and persistent inflammation. Effective regeneration of the intestinal epithelium relies on the maintenance of stemness and controlled ROS homeostasis—processes that are disrupted in IBD. Lipid droplets (LDs), endogenous lipid-storage organelles, have recently been recognized as active regulators of lipid metabolism, antioxidant defense, and stem cell homeostasis. Through their ability to buffer reactive oxygen species and supply metabolic substrates for regeneration, LDs may contribute to the recovery of intestinal function under inflammatory conditions.
Based on these biological properties, this study explored LDs as multifunctional bioinspired materials with therapeutic potential for IBD. LDs were isolated from murine adipose tissue and further processed into uniform nano-sized lipid droplets (nLDs) with improved stability and usability. The effects of nLDs were first evaluated in normal intestinal organoids under different treatment conditions (PRE or POST) to determine how exposure timing influences growth dynamics and cellular responses. nLD treatment promoted proliferation and mucus-associated functions, while in injured organoids exposed to lipopolysaccharide (LPS) and hydrogen peroxide (H₂O₂), nLDs alleviated oxidative damage and preserved barrier integrity. Furthermore, nLDs were engineered to encapsulate dexamethasone (nLD–DEX), integrating the intrinsic regenerative effects of nLDs with the anti-inflammatory activity of the drug. In vitro nLD–DEX modulated macrophage polarization and cytokine secretion, and in vivo administration reduced oxidative stress and restored mucosal architecture in a dextran sulfate sodium (DSS)-induced colitis model.
Overall, this work highlights the dual role of nLDs as both biologically active entities and natural drug carriers. By harnessing their inherent antioxidant and regenerative properties, along with their engineering flexibility for drug loading, this study establishes nLDs as a promising therapeutic platform for IBD and potentially other inflammation-related diseases.

국문 초록 (Abstract)

염증성 장질환(Inflammatory bowel disease, IBD)은 산화적 스트레스, 상피 장벽 기능 저하, 그리고 만성적인 염증을 특징으로 하는 질환이다. 장 상피의 효과적인 재생은 줄기세포성(stemness) 유지와 적절한 활성산소종 균형에 의존하며, 이러한 과정은 IBD에서 쉽게 붕괴된다. 최근 지질방울(lipid droplet, LD)은 단순한 지방 저장소가 아니라, 지질대사 조절, 항산화 방어, 줄기세포 항상성 유지에 관여하는 능동적 세포소기관으로 주목받고 있다. LD는 활성산소종(reactive oxygen species)을 완충하고 재생에 필요한 대사 기질을 공급함으로써 염증성 환경에서 장 기능 회복에 기여할 수 있다.
이러한 생물학적 특성을 바탕으로, 본 연구에서는 LD를 다기능성 생체유래 치료소재로 활용하여 IBD 치료 가능성을 탐색하였다. LD는 생쥐의 지방조직으로부터 분리한 뒤, 안정성과 조작성 향상을 위해 가공하여 균일한 나노 크기의 지방방울(nano LD, nLD)을 제작하였다. 이후 nLD의 효과를 정상 장 오가노이드에 적용하여, 처리 시점에 따른 성장 반응과 기능적 변화를 비교함으로써 오가노이드 발달 과정에서의 생리적 영향을 평가하였다. 그 결과, nLD 처리군은 오가노이드의 증식과 점액 관련 기능을 촉진하였으며, LPS와 H₂O₂로 손상된 오가노이드에서는 산화 손상을 완화하고 장벽 무결성을 유지하였다. 또한 nLD에 덱사메타손(dexamethasone)을 탑재하여 nLD–DEX를 제작함으로써, nLD의 고유한 재생 촉진 효과와 약물의 항염증 작용을 결합하였다. nLD–DEX는 in vitro에서 대식세포의 M2형 극화를 유도하고 사이토카인 분비를 조절하였으며, in vivo에서는 DSS로 유도한 대장염 마우스 모델에서 산화 스트레스를 감소시키고 점막 구조를 부분적으로 회복시켰다.
종합하면, 본 연구는 nLD가 생물학적 활성을 지닌 치료 인자이자 천연 약물 전달체로서의 이중 기능을 수행함을 보여준다. nLD의 항산화 및 재생 촉진 능력과 약물 적재를 위한 공학적 유연성을 결합함으로써, 본 연구는 IBD를 비롯한 다양한 염증성 질환 치료를 위한 nLD 기반 치료 전략의 가능성을 제시한다.

목차 (Table of Contents)

• 1. INTRODUCTION 1
• 1.1. Inflammatory bowel disease (IBD) 1
• 1.2. Role of LD in the stemness of intestinal stem cells (ISCs) 4
• 1.3. LD as a scavenger of free fatty acids (FFAs) and reactive oxygen species (ROS) 7
• 1.4. Application potential of LDs as functional biomaterials 9
• 1.5. The scope of this study 11
• 2. MATERIALS AND METHODS 14
• 2.1. Animals 14
• 2.2. Preparation of nLD from mouse inguinal white adipose tissues 15
• 2.3. Characterization of nLD 16
• 2.4. Cell culture 16
• 2.5. Isolation and culture of mouse small intestinal organoids 17
• 2.6. Western blot 18
• 2.7. Uptake of nLD into cell and organoid 19
• 2.8. ROS and FFA scavenging test 20
• 2.9. Cell viability and proliferation test 22
• 2.10. Quantitative polymerase chain reaction (qPCR) 23
• 2.11. Immunocytochemical staining 25
• 2.12. Caspase-Glo® 3/7 3D Assay 26
• 2.13. Malondialdehyde (MDA) assay 27
• 2.14. Analysis of macrophage polarization 28
• 2.15. Enzyme-linked Immunosorbent Assay (ELISA) 29
• 2.16. Dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model 30
• 2.17. Myeloperoxidase (MPO) assay 31
• 2.18. Histological analysis 32
• 2.19. Statistical analysis 32
• 3. RESULTS 33
• 3.1. Characterization and in vitro functional evaluation of adipose tissue-derived lipid droplets (LDs) 33
• 3.1.1. Physicochemical properties of adipose tissue-derived nLDs 33
• 3.1.2. Cellular uptake of nLDs 38
• 3.1.3. In vitro functionality of nLDs 41
• 3.2. Regulation of stemness and differentiation in intestinal organoids following nLD uptake 46
• 3.2.1. Distinct delivery modes of nLDs into intestinal organoids 46
• 3.2.2. nLD-mediated enhancement of organoid growth and proliferation 49
• 3.2.3. Transcriptional regulation of intestinal cell-type markers 52
• 3.2.4. Immunofluorescent validation of intestinal cell-type markers 55
• 3.3. Enhancement of proliferation and partial ROS attenuation by nLD treatment 58
• 3.3.1. Establishment of the intestinal injury model 58
• 3.3.2. Enhancement of proliferation and partial ROS attenuation by nLD treatment 61
• 3.3.3. Recovery of epithelial lineage and ferroptosis-related markers 66
• 3.3.4. Immunofluorescence validation of barrier restoration 70
• 3.4. nLD as an efficient drug delivery carrier 72
• 3.5. nLD treatment attenuates intestinal inflammation in the IBD mouse model 78
• 3.5.1. Experimental design and macroscopic recovery 78
• 3.5.2. Restoration of epithelial regeneration 81
• 3.5.3. Suppression of neutrophil infiltration and inflammatory cytokine production 83
• 3.5.4. Histological evaluation of mucosal recovery 86
• 4. DISCUSSION 88
• 4.1. Summary of the study 88
• 4.2. Limitations and future perspectives 90
• 4.2.1. Need for mechanistic elucidation 90
• 4.2.2. Need for in vivo stability and delivery analysis 91
• 4.2.3. Model limitations and translational applicability 92
• 4.3. Significance of the study 93
• 5. CONCLUSION 95
• 6. REFERENCES 97
• ABSTRACT IN KOREAN 107