Investigation of LNP-BDNF mRNA and Cargocyte-IL-10 as Dual Modalities for In Vitro Models of Ischemic Stroke = 허혈성 뇌졸중 In Vitro 모델에서의 LNP-BDNF mRNA및 Cargocyte-IL-10의 이중 치료 효과 연구

다국어 초록 (Multilingual Abstract)

Ischemic stroke is one of major causes of mortality and long-term neurological disability all over the world, especially resulting from the sudden blockage of cerebral blood flow. The deprivation of oxygen and glucose triggers continuous pathological events, including metabolic disorders, excitotoxicity, oxidative stress, and apoptosis, ultimately leading to neuronal loss and deficient neurological function. Current therapeutic strategies, likes thrombolytic agents and neuroprotective drugs, are limited by narrow treatment windows, low systemic bioavailability, and poor penetration for blood–brain barrier (BBB), highlighting the need for substitute approaches that can effectively address multiple pathological mechanisms. Messenger RNA (mRNA)-based therapeutics with gene delivery and cell-mediated cytokine delivery have developed as new strategies for various diseases. Brain-derived neurotrophic factor (BDNF) is important for promoting neuronal survival, maintaining synaptic integrity, and facilitating functional recovery. Also, interleukin-10 (IL-10) is a potent anti-inflammatory cytokine which can attenuate neuroinflammation caused by ischemia. In this study, we investigated two kinds of therapeutic agents using in vitro ischemic stroke models induced by oxygen-glucose deprivation (OGD). Treatment with lipid nanoparticle-encapsulated BDNF mRNA (LNP-BDNF mRNA) caused increased intracellular BDNF levels significantly, enhanced neuronal viability, and reduced apoptotic cell death, demonstrating a cellular protective effect. In addition, Cargocyte-mediated IL-10 (Cargocyte-IL-10) delivery was used as a cell-based carrier system to enhance the stability and intracellular availability of IL-10. It could suppress effectively the expression of pro-inflammatory cytokines, thereby alleviating inflammation-induced cellular damage. These findings indicate that LNP-BDNF mRNA and Cargocyte-IL-10 induce individual protective effects against ischemic injury, with LNP-BDNF mRNA promoting neuronal survival and Cargocyte-IL-10 leading targeted anti-inflammatory action. Overall, this study highlights the therapeutic potential of both strategies and suggests the various applications of RNA-based and cell-based delivery platforms in the treatment of ischemic stroke.

국문 초록 (Abstract)

허혈성 뇌졸중은 전 세계적으로 주요한 사망 원인이자 장기적인 신경학적 장애를 유발하는 질환으로, 뇌혈류가 급격히 차단되면서 발생한다. 산소와 포도당 공급의 결핍은 대사 이상, 흥분독성, 산화 스트레스, 세포자멸사와 같은 병리적 연쇄반응을 유발하며, 결국 비가역적인 신경세포 손실과 기능 장애로 이어진다. 현재 임상에서 사용되는 혈전용해제 및 신경보호제는 제한된 치료 시간, 낮은 전신 생체이용률, 혈액–뇌 장벽(BBB) 투과성의 한계로 인해 효용성이 제한적이며, 이에 따라 다양한 병태기전을 동시에 조절할 수 있는 새로운 치료 전략이 필요하다. 최근 들어 메신저 RNA(mRNA) 기반 치료제와 세포 기반 사이토카인 전달 플랫폼이 신경 재생 치료제로 주목받고 있다. 뇌유래신경영양인자(BDNF)는 신경세포 생존과 시냅스 유지, 기능 회복을 촉진하며, 인터루킨-10(IL-10)은 강력한 항염증 사이토카인으로 허혈 후 발생하는 신경 염증 반응을 완화하는 데 중요한 역할을 한다. 본 연구에서는 산소–포도당 결핍(OGD)으로 유도한 in vitro 허혈성 뇌졸중 모델을 이용하여 두 가지 독립적 치료 전략의 효과를 평가하였다. LNP-BDNF mRNA처리 결과, 세포 내 BDNF 발현이 유의하게 증가하였으며, 신경세포 생존율이 향상되고 세포자멸사가 감소하였다. 이는 LNP-BDNF mRNA가 신경영양학적인 역할을 통해 허혈 스트레스 상황에서 신경세포 생존을 촉진함을 보여준다. 한편, Cargocyte-IL-10은 세포 기반 운반체를 이용하여 IL-10의 안정적인 전달과 세포 내 가용성을 높이는 방식으로 적용되었으며, 염증성 사이토카인의 발현을 억제하여 염증에 의한 2차적 세포 손상을 효과적으로 완화하였다. 이러한 결과는 LNP-BDNF mRNA가 주로 신경세포 생존을 강화하고, Cargocyte-IL-10이 항염증 작용을 통해 허혈성 뇌손상으로부터 세포를 보호함을 시사한다. 따라서 본 연구는 두 가지 치료 전략이 각각 독립적으로 허혈성 뇌졸중 병태생리에 대응할 수 있는 잠재력을 지님을 보여주며, 향후 새로운 뇌졸중 치료제 개발을 위한 중요한 근거를 제공한다.

목차 (Table of Contents)

• Chapter 1. INTRODUCTION 1
• 1.1 Ischemic Stroke 1
• 1.2 Lipid Nanoparticle-Mediated mRNA Delivery System 2
• 1.3 Brain-derived Neurotrophic Factor (BDNF) 4
• 1.4 Cargocyte 5
• 1.5 Interleukin-10 (IL-10) 6
• Chapter 2. MATERIALS & METHODS 8
• 2.1 Cell Culture 8
• 2.1.1 PC12 8
• 2.1.2 RAW264.7 9
• 2.1.3 hTERT immortalized-Adipose derived Mesenchymal Stem Cell (hT-MSC) 9
• 2.1.4 hTERT-immortalized IL-10-overexpressing adipose-derived Mesenchymal Stem Cell (IL-10-MSC) 10
• 2.2 Primary Culture of Cortical Neuron 10
• 2.3 Oxygen-Glucose Deprivation (OGD) Model 12
• 2.4 Lipid Nanoparticle (LNP)-mediated mRNA Transfection 13
• 2.5 Enucleation for Cargocyte Production 13
• 2.5.1 Ficoll Solution Preparation 13
• 2.5.2 Ultracentrifugation and Layer Isolation 15
• 2.6 Preparation of Conditioned Media (CM) 16
• 2.7 Western Blotting 17
• 2.7.1 Protein Extraction and Quantification 17
• 2.7.2 SDS-PAGE and Immunodetection 18
• 2.8 LPS-induced Inflammatory Response 18
• 2.9 Transwell assay 19
• 2.10 Cell viability assay 20
• 2.10.1 Treatment with Recombinant Human BDNF Protein
• 2.10.2 BDNF-Conditioned Media (BDNF-CM) Application
• 2.10.3 Assessment of Cell viability via MTT assay
• 2.11 Immunocytochemistry (ICC) 22
• 2.12 Flow Cytometric Analysis (FACS) 24
• 2.13 Enzyme-Linked Immunosorbent Assay (ELISA) 25
• 2.13.1 Human/Mouse BDNF ELISA
• 2.13.2 Human Interleukin-10 ELISA
• 2.13.3 Human TNF-α ELISA
• 2.14 Statistical Analysis 27
• Chapter 3. RESULT 28
• 3.1 Optimization of Oxygen-Glucose Deprivation (OGD) exposure duration and cytotoxicity assessment of LNP-BDNF mRNA in PC12 cells 28
• 3.2 Dose-dependent therapeutic response of LNP-BDNF mRNA under Ischemic condition 30
• 3.3 Anti-apoptotic effects of LNPBDNF mRNA in PC12 cells 32
• 3.4 Optimization of OGD exposure duration in Primary Cortical Neurons 34
• 3.5 Protective effects of LNPBDNF mRNA in Primary Cortical Neurons under Ischemic condition 36
• 3.6 Establishment of Cargocyte derived from hT-MSCs 40
• 3.7 Cytoprotective and anti-inflammatory effects of Cargocyte-IL-10 42
• Chapter 4. DISCUSSION 69
• References 72
• 논문요약 82