스테롤 유도체 기반의 양이온성 지질 조합에 의한 유전자 전달 효율 증가 = Enhancement of gene delivery efficiency by the combination of sterol derivative based cationic lipids

국문 초록 (Abstract)

콜레스테롤은 생분해성, 생체 적합성, 함입 효율 개선 등의 장점이 있어 유전자 전달체의 일부로 많은 연구가 있어왔으며, 콜레스테롤 기반의 양이온성 리포솜을 활용한 유전자 전달체도 널리 사용되고 있다. 최근 여러 연구에서 베타-사이토스테롤이 물리 화학적 특성 차이에 의해 세포내 전달과 엔도솜 탈출을 촉진하여 콜레스테롤보다 유전자 전달을 향상시킨다는 것이 입증되었다. 다른 연구에서 지질나노입자에 콜레스테롤의 25% 혹은 50%를 7α-하이드록시콜레스테롤로 대체하여 mRNA 전달을 개선할 수 있음을 확인했다.
본 연구에서는 기존에 합성된 스테롤 기반의 양이온 지질로 세 가지 유형의 양이온 리포솜을 형성하였다. 각각의 리포솜은 콜레스테롤 기반의 양이온성 지질으로 구성되거나(CCL), 베타-사이토스테롤 기반의 양이온성 지질으로 구성되거나(SCL) 두 가지의 양이온성 지질을 동일한 몰 비로 구성되었다(CCL+SCL). CCL+SCL은 CCL, SCL 보다 크기가 크고 제타전위가 높은 물리화학적 특성을 나타내었다. CHO-K1세포와 HeLa 세포에서 CCL+SCL은 plasmid DNA와 mRNA의 전달에서 CCL, SCL보다 우월한 전달 효율을 보여주었고, CCL+SCL 리포솜 형성을 위한 co-lipid로서 DOPE는 DSPC, DPPC, DOPC 등 다른 지질보다 높은 유전자 전달 효율을 나타내었다. 유세포분석기로 측정한 세포내 전달 실험에서 CHO-K1세포와 HeLa 세포에서 CCL+SCL은 CCL, SCL보다 빠른 세포내 전달과 속도를 보여주었다. 공초점 현미경으로 측정한 엔도솜 탈출 측정에서도 CCL+SCL은 CCL, SCL보다 높은 엔도솜 탈출효율을 보여주었다.
결론적으로, 본 연구는 CCL+SCL은 전달체의 크기와 제타전위 등의 물리화학적 특성에서 CCL, SCL 등과 차이가 있었으며, 세포내 전달과 엔도솜 탈출의 증가 등으로 plasmid DNA와 mRNA의 유전자 전달을 향상시킴을 확인하였다.

다국어 초록 (Multilingual Abstract)

The CCL+SCL enhanced gene delivery efficiency of plasmid DNA and mRNA prominently, although SCL also showed better transfection efficiency of plasmid DNA compared to CCL. It was consistent with several recent studies using β-sitosterol. Due to modified physiochemical properties of LNP affected by additional C-24 ethyl group at alkyl chain of β-sitosterol, LNP that contains β-sitosterol has shown improved the encapsulation ability, cellular uptake, and endosomal escape than LNP containing cholesterol [23]. LNP composed of β-sitosterol improved mRNA delivery efficiency than using cholesterol [21, 22, 25]. It was also a similar result that the substitution of 25% and 50% 7α- hydroxycholesterol that has additional hydroxyl groups for normal cholesterol in LNP enhanced mRNA delivery [26]. CCL+SCL was larger and showed more positive zeta potential than other liposomes, and these characteristics could induce increased transfection efficiency compared to other liposomes.
Transfection efficiencies according to the kinds of helper lipids and the weight ratios of liposome and nucleic acids were investigated. Transfection efficiency of CCL+SCL according to neutral helper lipids was enhanced in the order of DOPE, DOPC, DPPC and DSPC. Among these helper lipids, DOPE is the only lipid having primary amine head, it can be the reason for the superior property in transfection efficiency like previous study [27, 28]. The weight ratio of CCL+SCL: plasmid DNA = 10:1 or 12:1 was the optimal weight ratio for the transfection of plasmid DNA in CHO-K1 and HeLa cells.
Since the cellular uptake of liposome is one of the early events of the gene delivery process, the percentages of cells containing liposomes one hour after transfection were measured by flow cytometry. CCL+SCL and SCL showed more cellular uptake compared to CCL in CHO-K1 and HeLa, and this higher uptake might the cause of efficient gene expression. It is the consistent result that LNP that contains β-sitosterol has shown improvement over LNP containing cholesterol [23].
To compare each liposome’s endosomal escape which is important event in gene delivery [29], the intracellular distributions of each liposome in CHO-K1 were observed by confocal microscopy. The co-localization ratios between liposome and lysosome were calculated through the merged images. There was no significant difference in the proportion of between CLL and SCL, while co-localization ratio of CLL+SCL is almost half compared with other liposomes. It demonstrated that the combination of cholesterol-based cationic lipids and β-sitosterol-based cationic lipid in composition of liposome improve endosomal escape like a previous study [21-23]. Although it is possible that the combination of sterol-derivatives-based cationic lipids in CCL+SCL may be causing morphological changes to the liposome that impact cellular uptake and endosomal escape [22], further investigations are needed to clarify the mechanism of this enhancement of gene delivery.
In conclusion, the combination of β-sitosterol based cationic lipid and cholesterol based cationic lipid in forming liposomes increased the particle size and zeta potential, and enhanced the efficiency of gene delivery by improving cellular uptake, and endosomal escape.

목차 (Table of Contents)

• Introduction 1
• Materials & Methods 3
• Results 9
• Discussion 26
• References 29
• Summary 33
• Korean abstract 35