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Milk-derived Extracellular Vesicle-based Delivery Platform for Inflammatory Bowel Disease Treatment

  • 박혜민 (아주대학교 일반대학원)

초록/요약

Milk-derived Extracellular Vesicle-based Delivery Platform for Inflammatory Bowel Disease Treatment Inflammatory bowel disease （IBD） is a chronic, relapsing disorder driven by a self-reinforcing cycle in which intestinal epithelial barrier disruption, excessive pro-inflammatory cytokine production, and oxidative stress amplify one another. Current therapies rely largely on systemic immunosuppression, which controls inflammation but does not directly restore the epithelial barrier and carries risks of systemic side effects and incomplete mucosal healing. An effective strategy therefore requires the simultaneous restoration of barrier integrity and suppression of inflammation, ideally delivered orally in a form that survives gastrointestinal transit and acts locally at the diseased mucosa. Extracellular vesicles （EVs） have emerged as promising carriers for such targeted delivery, but cell-derived vesicles are limited by low scalability and poor stability in the harsh gastrointestinal environment. To address this, we developed milk-derived extracellular vesicles （MEVs） loaded with miR-200b as an orally administrable delivery platform. We reasoned that the rigid, sphingomyelin-rich lipid bilayer of MEV would protect the miRNA cargo during gastrointestinal transit, while miR-200b would act on the miR-200/zinc finger E-box-binding homeobox 1 （ZEB1） axis to counter epithelial-to-mesenchymal transition （EMT） and barrier loss. MEV were isolated from bovine milk, characterized as extracellular vesicle-like particles, and shown to retain their inte grity under simulated gastrointestinal conditions far better than cell-derived vesicles, confirming their suitability as an oral carrier. Endogenous cargo was then depleted and miR-200b was reloaded by electroporation to generate miR-200b-enriched vesicles （miRELV）, with vesicle size and morphology preserved throughout. Functionally, miRELV suppressed EMT and restored tight junction integrity in oxidatively stressed intestinal epithelial cells, thereby recovering epithelial barrier function more effectively than unmodified MEV. miRELV likewise suppressed pro-inflammatory responses in macrophages more strongly than MEV. In a dextran sulfate sodium-induced colitis model, oral administration of MEV and miRELV alleviated disease severity and preserved intestinal barrier integrity, with tight junction proteins maintained in vivo. Collectively, this study demonstrates that miR-200b-loaded milk-derived extracellular vesicles act simultaneously on the barrier-inflammation axis of IBD and provide a biocompatible, scalable, and gastrointestinally stable oral platform. These findings highlight the potential of engineered dietary vesicles as a next-generation oral therapeutic strategy for IBD and more broadly for the targeted delivery of nucleic acid therapeutics to the intestine. Keywords: Inflammatory bowel disease, Milk-derived extracellular vesicles engineering, miR-200b, Oral delivery, Intestinal epithelial barrier restoration

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초록/요약

염증성 장질환은 장 상피 장벽 손상, 과도한 전염증성 사이토카인 생성, 산화 스트레스가 서로를 증폭시키는 자기강화 순환으로부터 진행되는 만성 재발성 질환이다. 현재의 치료 전략은 주로 전신 면역억제에 의존하는데, 이는 염증을 조절하기는 하나 상피 장벽을 직접 회복시키지 못하며 전신 부작용과 불완전한 점막 치유의 위험을 동반한다. 따라서 효과적인 치료를 위해서는 장벽 무결성 회복과 염증 억제를 동시에 달성하되, 위장관을 통과하여 살아남아 병변 점막에 국소적으로 작용할 수 있는 경구 형태의 전달 전략이 요구된다. 세포외소포는 이러한 표적 전달을 위한 유망한 운반체로 주목받고 있으나, 세포 유래 소포는 낮은 확장성과 가혹한 위장관 환경에서의 불안정성이라는 한계를 가진다. 이러한 한계를 극복하기 위해 본 연구에서는 miR-200b를 탑재한 우유 유래 세포외소포（MEV）를 경구 투여형 전달 플랫폼으로 개발하였다. 스핑고마이엘린이 풍부하여 강성이 높은 MEV의 지질 이중층은 위장관을 통과하는 동안 miRNA 화물을 보호하고, miR-200b/ZEB 축에 작용하여 상피-중간엽 전환（EMT）과 장벽 소실에 대응할 것으로 가정하였다. MEV는 소 우유로부터 분리하여 세포외소포 입자임을 확인하였으며, 모의 위장관 조건에서 세포 유래 소포보다 입자 무결성을 훨씬 잘 유지하여 경구 운반체로서의 적합성을 입증하였다. 이후 내인성 화물을 제거하고 전기천공법으로 miR-200b를 재탑재하여 miR-200b가 풍부한 소포（miRELV）를 제작하였으며, 이 과정 전반에서 소포의 크기와 형태는 유지되었다. 기능적으로 miRELV는 산화 스트레스를 받은 장 상피세포에서 EMT를 억제하고 밀착연접 무결성을 회복시킴으로써 미변형 MEV보다 상피 장벽 기능을 더욱 효과적으로 회복시켰으며, 이러한 추가적 이점은 소포 자체의 항산화 활성이 아니라 miR-200b에서 기인함을 확인하였다. 또한 miRELV는 대식세포에서 전염증성 반응을 MEV보다 더 강하게 억제하였으며, 대장염 마우스 모델에서 MEV와 miRELV의 경구 투여는 질병 중증도를 완화하고 밀착연접 단백질이 생체 내에서 보존되어 장벽 무결성을 유지하였다. 종합하면, 본 연구는 miR-200b를 탑재한 MEV가 염증성 장질환의 장벽-염증 축에 동시에 작용함을 보여주며, 생체적합성과 확장성, 위장관 안정성을 갖춘 경구 전달 플랫폼을 제공한다. 이러한 결과는 공학적으로 가공된 식품 유래 소포가 염증성 장질환에 대한 차세대 경구 치료 전략으로서, 나아가 장으로의 핵산 치료제 표적 전달을 위한 플랫폼으로서 갖는 잠재성을 제시한다. 키워드: 염증성 장질환, 우유 유래 세포외소포, miR-200b, 경구 전달, 장 상피 장벽 회복

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목차

ABSTRACT I
1. INTRODUCTION 1
1.1 Inflammatory bowel disease 1
1.2 Intestinal inflammation and epithelial barrier dysfunction 3
1.3 Milk-derived extracellular vesicles as oral delivery platform 7
1.4 miR-200b as a therapeutic cargo for epithelial barrier recovery 9
1.5 Objectives of this study 11
2. MATERIALS AND METHODS 13
2.1 Cell culture 13
2.1.1 Caco-2 cell culture 13
2.1.2 THP-1 cell culture 13
2.2 Isolation and characterization of MEV 14
2.2.1 Isolation and purification of MEV 14
2.2.2 Nanoparticle tracking analysis (NTA) 15
2.2.3 Cryo-transmission electron microscopy (Cryo-TEM) 15
2.2.4 Single-EV tetraspanin profiling 16
2.2.5 Lipidomics analysis 16
2.3 Gastrointestinal stability assessment of MEV 18
2.4 Cargo depletion and therapeutic miRNA loading 19
2.4.1 Endogenous cargo depletion: preparation of MELV 19
2.4.2 Therapeutic miRNA loading into MELV: preparation of miRELV 19
2.4.3 Quantification of miRNA loading in miRELV 21
2.5 Cellular uptake of MEV in Caco-2 cells 22
2.6 Cell viability assessment in Caco-2 cells 23
2.7 miR-200b target gene expression in Caco-2 cells 24
2.8 Intracellular ROS measurement by DCF-DA assay 25
2.9 Evaluation of Caco-2 monolayer barrier function 26
2.10 Inflammatory cytokine expression in M1 macrophages 27
2.11 Flow cytometric analysis of M1-associated surface markers 28
2.12 Animal study in a DSS-induced colitis model 29
2.12.1 In vivo intestinal uptake of orally administered MEV 29
2.12.2 Animal model and experimental design 29
2.12.3 Disease activity index assessment 30
2.12.4 Histology analysis 31
2.13 Statistical analysis 32
3. RESULTS 36
3.1 MEV were successfully isolated from bovine milk and characterized as extracellular vesicle-like particles 36
3.2 Lipidomics analysis revealed the membrane lipid composition of MEV 39
3.3 MEV were stable under simulated gastrointestinal conditions and reached the intestine after oral administration 43
3.4 miR-200b-loaded miRELV were prepared from cargo-depleted MEV 48
3.5 MEV-based formulations showed no significant cytotoxicity in Caco-2 cells 53
3.6 miRELV suppressed EMT markers and restored tight junction markers in oxidatively stressed Caco-2 cells 55
3.7 miRELV promoted epithelial barrier recovery and attenuated intracellular ROS in oxidatively stressed Caco-2 cells 57
3.8 miRELV suppressed pro-inflammatory gene expression and M1 surface markers in THP-1-derived M1 macrophages 62
3.9 Oral administration of MEV and miRELV accelerated recovery and prevented colon shortening in DSS-induced colitis 65
3.10 MEV and miRELV suppressed intestinal COX-2 expression and preserved tight junction proteins in vivo 68
4. CONCLUSION 70
REFERENCES 74
ABSTRACT IN KOREAN 79

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