Cationic drug-derived lipids as delivery systems of nucleic acids

다국어 초록 (Multilingual Abstract)

siRNA has a great potential as therapeutics. However, difficulty of delivery into target cells has hampered development of siRNA as therapeutics. Therefore, many vectors as siRNA delivery systems have been developed. Among various delivery systems, many lipid-based carriers, a major category of the non-viral vectors, have been developed due to its high transfection efficiency. In this study, we synthesized hydrophobically modified compound X (compound Y) as a delivery system of siRNA via conjugation of compound X with oleic acid. Compound X is an antineoplastic agent used in the treatment of certain types of cancer. Uptake efficiency of siRNA mediated by compound X is greatly enhanced by hydrophobic modification of compound X. A fluorescent double-stranded RNA was used to visualize cellular uptake of siRNA by compound Y. Knock-down of mRNA expression in KB cells was tested after delivery of survivin-specific siRNA using compound Y by RT-PCR. A combined anticancer effect of compound Y and Mcl-1 specific siRNA was shown in A375 human melanoma cells and KB human epithelial carcinoma cells in vitro and KB tumor xenograft mice in vivo. These results suggest a novel concept of combined cancer therapy in which siRNA delivered by anticancer drug has a great potential as anticancer therapeutics.

국문 초록 (Abstract)

siRNA 는 치료제로서 매우 큰 잠재성을 가지고 있다. 그러나 표적 세포로의 전달이 어렵기 때문에 치료제로서의 개발에 장애가 되어왔고 따라서 siRNA 수송체 개발을 위한 수많은 연구가 진행되어왔다. 여러가지 siRNA 수송체 중에서도 비바이러스성인 지질 기반의 수송체는 높은 전달 효율로 인하여 많은 연구가 이루어져왔다. 본 연구에서는 항암제인 compound X 와 올레익산을 에스터 결합으로 접합하여 소수성으로 수식된 compound X 를 siRNA 수송체로서 사용하였다. compound X 는 항암제로서 여러가지 암의 치료에 사용되고 있다. Compound X 를 소수성으로 수식함으로써 siRNA 의 암세포로의 전달 효율이 매우 높아졌으며 이를 형광으로 표지된 double-stranded RNA 로 확인하였다. Survivin 에 특이적인 siRNA 를 소수성으로 수식된 compound X 로 KB 암세포주에 전달하여 mRNA 의 발현 억제를 확인하였다. 또한 Mcl-1 에 특이적인 siRNA 를 소수성으로 수식된 compound X 로 A375, KB 세포주에 전달하거나 KB 쥐 종양 모델에 주입하여 상승 된 항암 효과를 확인하였다. 이러한 결과들은 항암제로 siRNA 를 전달하여 더욱 상승 된 항암 효과를 보일 수 있는 새로운 개념의 항암 치료제 개발을 제안한다.

목차 (Table of Contents)

• INTRODUCTION = 1
• MATERIALS AND METHODS = 7
• 1. Materials = 7
• 2. Chemical synthesis of compound Y = 8
• 2.1. Boc-protecting reacion of compound X = 8
• 2.2. Coupling reaction of oleic acid and Boc-protected compound X = 9
• 2.3. Deprotecting reaction of Boc-protected dioleoyl-compound X = 10
• 3. Complex compound Y with siRNA and particle size measurements = 12
• 4. Gel retardation assay = 14
• 5. Compound Y mediated siRNA cellular uptake study by fluorescent microscopy = 15
• 6. Reverse transcription polymerase chain reaction (RT-PCR) = 17
• 7. MTT cell viability assay = 19
• 8. In vivo evaluation of compound Y and siMcl-1 in tumor bearing mice = 21
• 9. Statistical analysis = 22
• RESULTS = 23
• 1. Synthesis of compound Y as a siRNA carrier = 23
• 2. Characterization of compound Y and siRNA complexes = 25
• 3. Cellular uptake of dsRNA delivered by compound Y = 31
• 4. RNAi effect of survivin-specific siRNA delivered by compound Y = 34
• 5. Anticaner effects of compound Y and Mcl-1 specific siRNA delivered by compound Y in vitro = 36
• 6. Anticaner effects of compound Y and Mcl-1 specific siRNA delivered by compound Y in vivo = 39
• DISCUSSION = 41
• REFERENCES = 45
• ABSTRACT IN KOREAN = 52