Development of Protein-decorated Lipid Nanoparticles as an Oral mRNA Delivery Platform = 단백질 표면개질 지질 나노입자의 경구 mRNA 전달 플랫폼 개발

다국어 초록 (Multilingual Abstract)

Development of protein decorated lipid nanoparticles as an oral delivery platform by. Kim min jeong Department of Bio & Fermentation Convergence, Graduate School, Kookmin University, Seoul, Korea Oral administration of nucleic acid therapeutics remains a challenging task due to the harsh physiological and enzymatic environment of the gastrointestinal tract (GIT). To overcome these barriers, a lipid nanoparticle (TOBP) platform decorated with a traptavidin-oleosin protein and bioitin was developed to enhance the stability and intestinal absorption of orally administered mRNA. The TOBP system was manufactured by introducing a biotin-PEG-biotin linker and a traptavidin-oleosin recombinant protein into conventional lipid nanoparticles (LNPs), forming a stable biotin-protein interface capable of protecting mRNA from degradation. Dynamic light scattering (DLS) and zeta potential analysis revealed that TOBP, containing 2 kDa PEG-biotin, exhibited optimal size distribution and surface stability under thermal and digestive stress. TEM and confocal microscopy observations confirmed that TOBP maintained a spherical morphology and preserved mRNA encapsulation, whereas unmodified LNPs showed aggregation and structural loss. In vitro studies using Caco-2 cells demonstrated that TOBP exhibited stronger mRNA expression and higher fluorescence intensity compared to conventional LNPs. Oral administration of TOBP in Drosophila melanogaster revealed enhanced stability in gut and partial recovery of lipid metabolism in trpγ¹ mutant flies, proving efficient mRNA delivery and biological functionality. In vivo imaging analyses in mouse models revealed that TOBPs exhibited enhanced mRNA expression in the intestine compared to LNPs. Intracellular mRNA uptake was confirmed via frozen sections. Collectively, these results demonstrate that TOBPs provide enhanced stability and mRNA expression efficiency compared to conventional LNPs. This platform presents a promising strategy for developing safe and effective oral mRNA delivery systems for future therapeutic application.

국문 초록 (Abstract)

경구 투여는 환자의 순응도를 높일 수 있는 가장 편리한 약물 전달 방식 중 하나이지만,
위장관의 복잡한 생리학적 환경과 소화효소로 인해 mRNA 의 안정성과 흡수율이 크게
제한된다. 본 연구에서는 이러한 한계를 극복하기 위해 단백질로 표면 개질 된 지질
나노 입자(Traptavidin–Oleosin–Biotin Particle, TOBP)를 설계하고, 경구 투여 시의
안정성과 발현 효율을 평가하였다. TOBP 는 기존의 지질 나노 입자(Lipid Nanoparticle,
LNP)에 biotin–PEG–biotin linker 와 traptavidin–oleosin 재조합 단백질을 도입하여
합성하였다. 물리화학적 분석 결과, 2 kDa PEG-biotin 을 포함한 TOBP 가 균일한 입자
크기와 우수한 표면 전하 안정성을 보였으며, 고온 및 소화환경에서도 구조를
안정적으로 유지하였다. TEM 이미지와 형광 이미지 분석에서도 TOBP 가 구형 구조를
유지하며 mRNA 를 효과적으로 보호하는 것이 확인되었다. 초파리 모델(Drosophila
melanogaster)에서는 장 조직 내에서 mRNA 발현과 함께 trpγ¹ 돌연변이 개체의
지질대사 회복 효과를 나타냈다. 또한 생쥐 모델에서 경구 투여 후 형광 이미징을 통해 TOBP 가 LNP 대비 mRNA 발현이 소장에서 더 강하게 나타나는 양상을 보였으며,
조직 절편 분석을 통해 장 상피세포 내 mRNA 흡수가 확인되었다. 이러한 결과는
TOBP 가 기존 LNP 대비 우수한 안정성 및 소장 흡수율을 가지며, 안전하고 효과적인
경구용 mRNA 전달 플랫폼으로 활용될 수 있음을 보여준다.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Materials & Methods 4
• 2.1 Expression and Purification of recombinant protein 4
• 2.2 Synthesis of Lipid nanoparticle (LNP) and Traptavidin–Oleosin–Biotin lipid nanoparticle (TOBP) 4
• 2.3 Dynamic light scattering 5
• 2.4 Zeta potential 5
• 2.5 TEM image 5
• 2.6 Cell culture 5
• 2.7 In Vitro Simulated Gastric Fluids and Simulated Intestine Fluid Assays 6
• 2.8 Fluorescence imaging 6
• 2.9 Experimental conditions and Drosophila strains 6
• 2.10 Imaging assay Hoechst 33342 staining 6
• 2.11 Imaging assay Fluorescence imaging 7
• 2.12 TAG level measurement 7
• 2.13 Nile red staining 7
• 2.14 Experimental conditions and Mouse models 8
• 2.15 Imaging assay IVIS imaging 8
• 2.16 Imaging assay Fluorescence imaging 8
• 2.17 Statistical analysis 8
• 3. Results and Discussion 9
• 3.1 Synthesis and Characterization of LNPs and Traptavidin-Oleosin-Biotin Lipid nanoparticle (TOBP) 9
• 3.2 Thermal and Digestive Stability of LNP and Traptavidin-Oleosin-Biotin Lipid nanoparticle (TOBP) 11
• 3.3 Evaluation of Expression Efficiency in Drosophila melanogaster Models 18
• 3.4 Trpɣ mutant phenotype rescue in Drosophila melanogaster mutant models 22
• 3.5 Evaluation of Expression Efficiency in Mouse Models 26
• 4. Conclusion 30
• 5. Reference 31
• 6. Abstract in Korean 34