Engineered milk-derived exosomes for safe and efficient sonodynamic cancer therapy

국문 초록 (Abstract)

초음파역학 항암 치료 (sonodynamic cancer therapy, SDT)는 기존 화학요법의 한계를 극복할 수 있는 새로운 치료 전략으로, 전신 독성을 최소화하면서 심부 종양을 표적 치료할 수 있다. 유기 초음파감작제 (sonosensitizer)의 낮은 수용성과 낮은 종양 특이성을 극복하기 위해 효율적인 세포 내 전달을 위한 다양한 합성 전달체가 개발되어 왔다. 그러나 이러한 합성 전달체는 생체 내 독성 및 면역원성과 같은 문제를 야기하는 경우가 많아, 고도의 생체적합성을 지닌 전달 시스템의 개발이 요구된다. 본 연구에서는 우유 유래 엑소좀 (MExo)을 초음파감작제 전달체로 활용하였다. MExo는 기존의 세포 유래 엑소좀에 비해 대량 생산이 용이하고 경제성이 뛰어나 생체적합한 전달체로서 높은 잠재력을 지닌다. 또한, 엑소좀 막에 diselenide 기반 링커를 도입하여 글루타치온 반응성 MExo를 개발함으로써, 초음파감작제의 효율적인 세포 내 전달을 구현하였다. 글루타치온 (glutathione, GSH) 반응성 엑소좀은 암 세포 내에 높은 농도로 존재하는 글루타치온에 의해 다이셀레나이드 링커가 분해되면서 약물이 방출되도록 설계되었다. 먼저, 인간 유방암 세포 (MCF-7)에서 Chlorin e6 (Ce6)가 적재된 우유 유래 엑소좀의 초음파역학 치료 효능을 평가하였다. 다이셀레나이드 링커가 결합된 MExo (DMExo)는 종양 환경과 유사한 GSH 농도 (~10 mM)에서 GSH 반응성이 아닌 엑소좀에 비해 향상된 약물 방출을 보였다. Ce6가 적재된 DMExo (Ce6-DMExo)를 MCF-7 세포에 처리했을 때, DMExo는 세포질 내에서 Ce6의 방출을 촉진하였다. 그 결과 초음파 (ultrasound, US) 조사 시 활성산소종 (reactive oxygen species, ROS) 생성이 증가하였고, MCF-7 세포에서 유의미한 세포 사멸이 유도되어 SDT를 위한 효과적인 전달체로서의 높은 가능성을 입증하였다. 다음으로, 교모세포종 (glioblastoma, GBM) 치료를 위한 SDT 전달체로서 MExo의 활용 가능성을 평가하였다. GBM 치료에서 혈액-뇌 장벽 (blood–brain barrier, BBB)은 주요한 장애 요소이므로, MExo의 BBB 투과 효율을 평가하였다. In vitro BBB transwell 모델을 이용하여 MExo, DMExo, 그리고 인간 배아 신장세포 (HEK-293T) 유래 엑소좀 (HEKExo)의 BBB 투과 효율과 세포 내 흡수를 비교하였다. Ce6-MExo는 자유 Ce6에 비해 GBM에서 유의하게 향상된 BBB 투과성과 세포 내 흡수를 보였다. 특히, MExo의 BBB 투과성은 HEKExo와 유사한 수준이었다. 또한 Ce6- DMExo와 Ce6-MExo는 유사한 BBB 투과성을 나타냈다. 그럼에도 불구하고, Ce6-DMExo는 GBM 세포 내에서 Ce6를 효율적으로 방출함으로써 가장 높은 초음파 독성 (sonotoxicity)을 보였다. 이러한 결과들은 Ce6-DMExo가 유기 초음파감작제의 낮은 세포 흡수율과 낮은 BBB 투과성이라는 한계를 극복할 수 있는 안전하고 효과적인 SDT 전달체로서 큰 잠재력을 지니고 있음을 보여준다.

다국어 초록 (Multilingual Abstract)

Sonodynamic cancer therapy is an emerging therapeutic strategy that can overcome the limitations of conventional chemotherapy through targeted treatment of deep-seated tumors while minimizing systemic toxicity. Various synthetic carriers have been developed for efficient intracellular delivery of organic sonosensitizers by overcoming low water solubility and poor tumor specificity. However, these synthetic carriers often pose challenges such as in vivo toxicity and immunogenicity, necessitating the development of highly biocompatible delivery systems. In this study, I utilized milk-derived exosomes (MExo) as sonosensitizer delivery vehicles. MExo possess high potential as biocompatible carriers due to their superior scalability and cost-effectiveness compared to conventional cell-derived exosomes. Additionally, I developed glutathione (GSH)-responsive MExo for efficient intracellular delivery of sonosensitizers by incorporating diselenide linkers into the exosome membranes. The GSH-responsive exosomes were designed to facilitate drug release within cancer cells via the degradation of diselenide linkers in response to the high concentration of GSH in cancer cells. First, I demonstrated the efficient sonodynamic therapy (SDT) efficacy of chlorin e6 (Ce6)-loaded MExo in human breast cancer cells (MCF-7). The diselenide linker-conjugated MExo (DMExo) exhibited enhanced drug release under tumor-relevant GSH concentrations (~ 10 mM) compared to non-GSH-responsive exosomes When Ce6-loaded DMExo (Ce6- DMExo) was added to In MCF-7 cells, DMExo facilitated the release of Ce6 in the cytoplasm. This led to enhanced generation of reactive oxygen species (ROS) upon ultrasound (US) irradiation and resulted in significant cell death in MCF-7 cells, highlighting its high potential as effective carriers for SDT. Next, I evaluated the feasibility of using MExo as SDT carriers for the treatment of glioblastoma (GBM). Since the blood-brain barrier (BBB) is the primary obstacle in GBM therapy, the BBB penetration efficiency of the MExo was assessed. The BBB permeation efficiency and intracellular uptake of MExo, DMExo, human embryonic kidney cell (HEK-293T)-derived exosomes (HEKExo) were compared using in vitro BBB transwell model Ce6-MExo exhibited significantly enhanced BBB permeation and cellular uptake in GBM compared to free Ce6. Notably, the BBB permeability of MExo was comparable to that of HEKExo. Moreover, Ce6-DMExo and Ce6- MExo showed similar BBB permeability. Notably, Ce6-DMExo exhibited the highest sonotoxicity owing to its efficient release of Ce6 in GBM cells. These studies demonstrate the potential of milk-derived exosomes as safe and effective SDT carriers that overcome the limitations of poor cellular uptake and low BBB permeability of organic sonosensitizers.

목차 (Table of Contents)

• Abstact i
• Table of Contents iii
• List of Tables vi
• List of Figures vii
• Chapter 1. Introduction 1
• 1.1. Research background 1
• 1.1.1. Sonodynamic therapy for noninvasive and selective treatment of tumors 1
• 1.1.2. Exosome-based drug delivery systems for enhanced bioavailability of sonosensitizers 2
• 1.1.3. Limitations of native exosomes 3
• 1.1.4. Blood-brain barrier (BBB): A major challenge in brain tumor therapy 4
• 1.1.5. Therapeutic potentials of milk-derived exosomes for BBB penetration 5
• 1.2. Research purpose 6
• Chapter 2. Glutathione-sensitive milk exosomes for safe and efficient sonodynamic cancer therapy 9
• 2.1. Materials 9
• 2.2. Methods. 9
• 2.2.1. Synthesis of diselenide-bearing lipid (DSe) 9
• 2.2.2. Isolation of milk derived exosomes (MExo) 10
• 2.2.3. Preparation and characterizations of Ce6-DMExo and Ce6-MExo 11
• 2.2.4. Evaluation of colloidal stability of Ce6-DMExo in physiological conditions & cancer intracellular conditions 11
• 2.2.5. In vitro drug release studies 12
• 2.2.6. Evaluation ROS generation in cell-free system 12
• 2.2.7. Cellular internalization analysis of T-Ce6 and T-Ce6-loaded exosomes 13
• 2.2.8. Evaluation of in vitro ROS generation 14
• 2.2.9. In vitro cytotoxicity evaluation 14
• 2.3. Results and Discussion 15
• 2.3.1. Synthesis of diselenide-bearing lipid (DSe) 15
• 2.3.2. Characterization of GSH-Sensitive MExo Loaded with a Sonosensitizer 15
• 2.3.3. Colloidal stability of Ce6-MExo under physiological conditions and cancer cytoplasm condition. 16
• 2.3.4. GSHsensitive drug release by Ce6-DMExo 17
• 2.3.5. ROS generation in Cell-free system upon US 18
• 2.3.6. Efficient cellular uptake and cytoplasmic drug release by GSH-sensitive exosomes 19
• 2.3.7. US-triggered intracellular ROS generation using Ce6-loaded exosomes 20
• 2.3.8. Low inherent toxicity of milk-derived exosomes 20
• 2.3.9. Glutathione-Sensitive Sonodynamic Cancer Therapy 21
• 2.4. Conclusion 32
• Chapter 3. Milk-derived exosomes as BBB-permeable nanocarriers for effective GBM treatment 33
• 3.1. Materials 33
• 3.2. Methods 33
• 3.2.1. Isolation of different types of exosomes 33
• 3.2.2. Assessment of BBB penetration 34
• 3.2.3. In vitro Cellular uptake in GBM 35
• 3.2.4. Enzyme-linked immunosorbent assay (ELISA) 35
• 3.2.5. In vitro cytotoxicity evaluation in BBB model 36
• 3.3. Results and Discussion 37
• 3.3.1. Intracellular uptake in GBM 37
• 3.3.2. BBB transcytosis of Ce6, Ce6-MExo, Ce6-HEKExo & Ce6-MExo, Ce6-DMExo 38
• 3.3.3. BBB Penetration followed by GBM Intracellular Uptake of Ce6-HEKExo, Ce6-MExo, Ce6 & Ce6-MExo, Ce6-DMExo 38
• 3.3.4. Transferrin receptor-mediated BBB transcytosis 39
• 3.3.5. Enhanced SDT effect by Ce6-DMExo in BBB model 40
• 3.4. Conclusions 48
• Reference 49
• 국문초록 57
• Acknowledgement 59