With high specificity and efficiency in target gene inhibition, small interfering RNAs (siRNAs) are promising tool for genomic therapeutics. To develop a siRNA delivery system with low cytotoxicity and high transfection efficiency, we prepared polyelectrolyte complex micelles (PECMs) of siRNA conjugated to poly(ethylene glycol) via a disulfide linkage (siRNA-PEG) using three kinds of condensing agents: amine-functionalized gold nanoparticles, cationic fusogenic peptide, Pluronic/poly(ethylenimine) nanocapsules.
Gold nanoparticles chemically modified with primary amine groups could form stable polyelectrolyte complexes through electrostatic interactions with negatively charged siRNA-PEG conjugates having a cleav-able di-sulfide linkage under reductive cytosol condition. The resultant core/shell type polyelectrolyte com-plexes surrounded by a protective PEG shell layer had a well dispersed nanostructure with a hydrodynamic diameter of 96.3 ± 25.9 nm, as determined by dynamic light scattering and transmission electron microscopy. Confocal laser scanning microscopy revealed that the nanosized polyelectrolyte complexes were efficiently internalized in human prostate carcinoma cells (PC-3 cells), and thus enhanced intracellular uptake of siRNA. Furthermore, the siRNA/gold complexes significantly inhibited the expression of a target gene within the cells without showing severe cytotoxicity. The current study demonstrated that positively charged gold nanoparticles could be potentially applied for intracellular delivery of siRNA.
Anionic siRNA-PEG conjugate and cationic KALA spontaneously formed nano-sized PECMs (< 200 nm) that have an inner core of charge neutralized siRNA/KALA complex surrounded by a PEG corona. VEGF siRNA was used to demonstrate VEGF sequence-specific gene inhibition in PC-3 cells. The extent of gene silencing was gradually increased with increasing nitrogen to phosphate (N/P) ratio and the concentra-tion of siRNA-PEG/KALA PECMs. These results suggest that the formulation of siRNA-PEG/KALA PECMs could be widely applied for intracellular delivery of various therapeutic siRNAs.
Pluronic/poly(ethylenimine) nanocapsules exhibiting a thermally reversible swelling/deswelling vol-ume expansion behavior were synthesized and used for an siRNA delivery nanocarrier as well as an effective endosome breaking agent. Pluronic/PEI NCs were in a collapsed state with an average size of 118.9 ± 15.3 nm at 37°C, but in a swollen state with that of 412.3 ± 83.2 nm at 15°C. The collapsed Pluronic/PEI NCs having a highly positive surface zeta potential value were utilized to load siRNA-PEG conjugate on the surface via electrostatic interactions. The Pluronic/PEI/siRNA-PEG nano-complexes were transfected to the cells at 37°C for efficient cellular uptake. The transfected cells were treated with a brief cold shock to induce an abrupt volume expansion of the NC within an endosome compartment to physically burst out the endosomal membrane. Far efficient gene silencing effects for both GFP and VEGF were observed after the cold shock treatment to the transfected cells.
Design and evaluation of novel siRNA structure are attractive and essential to increase its potency for biomedical applications. Here three kinds of chemically modified siRNA conjugates were demonstrated: siRNA-PEG multi-block copolymers, dual gene targeted multi-siRNA, and siRNA-PLGA conjugates. One of major missions of siRNA carrier is to protect vulnerable siRNA from environmental physical and biochemical attack. Di- and tri-block copolymers of siRNA with PEG were developed and assessed for enhanced stability and gene silencing effect. A single strand sense siRNA (sssiRNA) and a single stranded antisense siRNA (sassiRNA) were conjugated with PEG via a disulfide linkage. A-B (A: PEG; B: double strand siRNA(dsiRNA)) type di-block copolymer and A-B-A type tri-block copolymer were successfully prepared by stoichiometic hybridization of sssiRNA-PEG conjugate with a single stranded antisense siRNA (sassiRNA) and sassiRNA-PEG conjugate, respectively. The resultant di- and tri-block copolymers of PEGylated dsiRNA were characterized by gel permeation chromatography and gel electrophoresis. The enhanced enzymatic stability of siRNA in di- and tri-block copolymers was examined by incubating them with serum proteins. Using cationic Solid Lipid Nanoparticles (SLN) as a model vector system, effects of PEGylation density of dsiRNA-PEG copolymer/SLN complexes on gene silencing and cellular uptake were investigated by fluorophotometer, confocal microscopy, and FACS analysis. Evaluation of optimal PEG density on the nanosized carrier using polymer-siRNA block copolymer system could be exploited as a crucial and fundamental platform study for the development of improved siRNA carrier.
In our previous study, chemically self-crosslinked and multimerized siRNA (multi-siRNA) structure inhibited the expression of a target gene more efficiently in vitro and in vivo than naked siRNA by forming polyelectrolyte nano-complexes with a cationic polymer. Here we suggest advanced multimerized siRNA system composed of two different siRNA sequences for the selective and efficient inhibition of two target gene simultaneously at one time. Two model siRNAs against VEGF and GFP gene were chemically cross-linked via cleavable disulfide bond and noncleavable linkage for the preparation of dual gene targeted multi-siRNA conjugates (DGT multi-siRNA) using new synthetic schemes. DGT multi-siRNA via cleavable linkage exhibited enhanced gene silencing compared to DGT multi-siRNA via noncleavable linkage as well as elicited negligible immune response. Significantly superior gene silencing effects in both GFP and VEGF gene by DGT multi-siRNA were observed in at the level of mRNA and protein level, compared to physical mixture of naked siRNA or single gene targeted multi-siRNA (SGT multi-siRNA), which allowed to decrease effective dose of siRNA. DGT multi-siRNAs against two therapeutic siRNAs, anti- Survivin and anti-Bcl-2 targeted siRNA, showed greatly elevated apoptotic effect, which was attributed to elevated extent of their intracellular uptake compared to other control siRNAs. This novel approach for co-concurrent suppression of silencing dual combinatorial therapeutic target genes simultaneously using cleavable multimeric siRNA structure can be potentially used as a robust siRNA therapeutic system for the improved potency without eliciting side-effects.
For their clinical applications, specialized delivery strategy for siRNAs should be designed to over-come their insufficient charge density and form more compact and stable complex with cationic carriers. Here, biodegradable and biocompatible PLGA was conjugated to 3` end of siRNA via a disulfide bond for the synthesis of siRNA-PLGA hybrid copolymers and their micelle formation. Dynamic light scattering and atomic force microscopy analysis demonstrated that siRNA-PLGA copolymers were spontaneously self-assembled to form a homogenous micellar structure in an aqueous environment because of their amphiphilic property. The formation of siRNA-PLGA micelle allowed superior intracellular uptake and enhanced gene silencing effect with low molecular weight linear polyethylenimine as a micelle shell crosslinker because of its stable and homogenous nanostructure, compared to naked siRNA. This novel micelle structure based on siRNA can be potentially used as an efficient siRNA delivery system for the improved target gene silencing.
소간섭 알엔에이 (small interfering RNA, siRNA)는 19개에서 22개 정도의 핵산으로 구성된 짧은 이중 나선의 RNA가닥으로 이들과 염기서열이 동일한 유전자의 mRNA(messenger RNA)를 표적으로 삼아 유전자 발현을 억제시키는 것이다. 그러나 siRNA를 유전자 치료제로 이용하기 위해서는 siRNA의 안정성과 세포 전달성을 향상시킬 수 있는 효과적이고 안전한 유전자 전달 시스템이 구축되어야 한다. 현재 많이 시도되고 있는 유전자 전달 방법으로 siRNA를 발현하는 바이러스성 전달체를 체내에 주입함으로서 효과적으로 표적 단백질의 발현을 저해하는 경우가 있으나, 반복투여에 의한 면역반응 가능성과 바이러스의 병원성에 대한 우려로 실제 임상에 적용하기에 큰 어려움이 있다. 따라서 독성이 적은 다양한 양이온성 고분자, 지질, 단백질 전달체 등을 이용하여 합성 siRNA를 이온결합을 통해 나노 입자로 제작하고 이를 체내에 적용하려는 연구가 활발히 진행되고 있다. 표적 세포 내로 siRNA를 전달하여 치료효과를 극대화 하기 위해서는 다음과 같은 조건을 충족시키는 전달 시스템의 개발이 필요하다. RNase 노출에 의해 분해되어 기능을 상실하기 쉬운 RNA의 단점을 보완해야 하고 세포 독성과 부작용을 최소화 하기 위해 표적 세포 내로 정확하게 전달되어야 하며 세포 내에 endocytosis를 통해 도입된 후에는 세포질에 존재하는 표적 mRNA와 결합하기 위해 엔도좀을 효과적으로 탈출하여야 한다.
본 연구에서는 앞서 설명한 조건을 고려한 효과적이고 안전한 유전자 전달 시스템을 구축하기 위해 기존에 그 효과가 검증된 siRNA-PEG 접합체의 새로운 전달체를 개발하고 PEG 외에 다양한 기능성 분자가 결합된RNA 접합체를 합성하여, 기존의 siRNA가 가지는 단점을 개선하고 표적 단백질 저해 효율을 증가시켰다.
siRNA-PEG 접합체는 siRNA와 친수성 고분자인 폴리에틸렌글리콜(PEG)을 분해 가능한 황황다리결합으로 연결된 것으로 양이온성 고분자와 이온결합시 나노복합체가 형성된다. 이 복합체는 중심부에 siRNA가 있고 그 주위를 둘러싼 PEG가 혈액 내에서 대식세포나 핵산분해효소의 공격으로부터 siRNA를 보호하는 역할을 한다. siRNA와 안정적으로 결합되고 독성이 적으며 세포 내 엔도좀을 효과적으로 탈출할 수 있는 다양한 전달체를 개발하였다. 표면을 아민기로 치환하여 양이온 성질을 가지는 금나노 입자에 siRNA-PEG를 전기적 인력으로 결합시킨 후 세포 내로 전달시켜 표적 단백질이 효과적으로 저해되는 것을 확인하였고 생체 적합성 fusogenic 펩타이드인 KALA를 사용하여 siRNA-PEG를 효율적이고 안전하게 세포 내로 전달하여 RNA 간섭효과를 관찰하였다. 마지막으로 저온 충격으로 엔도좀을 효과적으로 탈출할 수 있는 온도 민감성 Pluronic/PEI 나노캡슐을 제작하여 이를 전달체로 이용함으로써 온도 변화에 따른 표적 단백질을 저해 효과를 관찰하였다.
PEG를 siRNA의 다양한 부위에 한 개 혹은 두 개를 결합시킨 siRNA-PEG 다중 블록 공중합 고분자 (multiblock copolymer)를 합성하여 PEG 밀도와 표적 단백질의 저해효과의 상관관계를 분석하였으며 이는 향후 PEG가 도입된 siRNA 나노입자의 연구에 중요한 지표로 활용될 것으로 기대된다. 또한 siRNA의 양이온성 전달체와의 이온결합을 증가시키기 위해 siRNA를 자가결합 시켜 siRNA 다중 접합체를 합성하였다. 두 종류 이상의 siRNA를 화학적 방법으로 결합시켜 복합 유전자를 표적하는 siRNA 다중 접합체를 제작하여 전하밀도와 유연성을 향상시키고 다양한 서열의 siRNA에 의한 각 서열 특이적/복합적 간섭 기능을 확인하였다. siRNA의 전하밀도를 증가시키기 위한 또 다른 방법으로 친수성 siRNA 말단에 생체적합성/분해성/소수성 고분자인 PLGA를 접합시켜 siRNA-PLGA micelle을 제작하였고, 독성이 적은 양이온성 고분자인 linear PEI를 micelle 표면에 전기적 인력으로 결합시킨 후 세포 내로 효과적으로 전달시켜 표적 단백질의 저해를 확인하였다.
다양한 기능성 분자를 화학적인 방법으로 결합시킨 siRNA 접합체를 이용한 본 연구에서는 유전자 전달체의 개발에만 국한되지 않은 새로운 형태의 유전자 전달 시스템을 개발함으로서 siRNA를 실제 임상에 적용 가능한 질병치료제로서 이용하는데 매우 유용한 역할을 할 것으로 기대된다.
