siRNA 전달을 위한 올리고 아르기닌-키토산 나노입자 제조 및 특성 분석

Abstract

siRNA는 다양한 질병의 치료를 위해 연구, 사용하고 있다. 그러나 siRNA 단독으로 사용하기에는 혈류 속에서 분해되고 신장에 의해 제거되어 세포 속으로 들어가기에 불충분하다. 이러한 한계를 극복하기 위해 안전성과 효율적인 유전자 전달능을 가진 전달체가 요구된다. 천연고분자로 알려진 키토산은 생체적합성과 생분해성이 뛰어나며 면역반응과 독성이 낮아 생체재료로 많이 사용되고 있다. 본 논문에서는 이러한 키토산에 아르기닌을 도입하여 특정 유전자의 억제 효과를 갖는 siRNA를 효율적으로 전달하기 위한 전달체를 연구하였다. 9R-키토산/siRNA 나노입자는 200 에서 600 nm 크기의 양전하는 띠는 나노입자로 유전자 전달체로써 적당한 크기를 지녔음을 확인했다. 또한, 혈청 단백질 내에서도 높은 안정성을 보여줬다. 혈청 단백질 존재 하에 진행됐던 9R-키토산 나노입자를 이용한 siRNA의 세포 내 유입과 좋은 유전자 억제 효과, 그리고 적은 독성은 비바이러스성 전달체로써 9R-키토산의 유용성을 증명해주었다.|Small interfering RNA (siRNA) has been exploited as a potential therapeutic for treatment of various diseases. However, the delivery of naked siRNA has raised several issues, including rapid enzymatic degradation, poor cellular uptake, limited stability, and low transfection efficiency. To overcome these limitations, siRNA has been combined with cationic polymers to enhance intracellular uptake and to increase stability. Chitosan is a naturally existing polysaccharide composed of glucosamine and N-acetylglucosamine residues, and can be derived by partial deacetylation of chitin obtained from crustacean shells. Chitosan is known to be biocompatible, low immunogenic, and non-toxic. Chitosan-based nanoparticles have been extensively used as a gene delivery carrier due to low toxicity and positively charged amino groups of chitosan. In this study, we hypothesized that an introduction of oligoarginine to chitosan could improve the ability of complex formation with siRNA. A peptide with nine repeating units of arginine (9R) was chemically coupled to the chitosan backbone and various characteristics of 9R-chitosan/siRNA nanoparticles were investigated. Their various physicochemical properties were characterized FT-IR, zetasizer, gel electrophoresis, FACS, and PCR. The mean diameter of 9R-chitosan/siRNA nanoparticles ranged from 210 nm to 600 nm, depending on the chitosan and 9R concentration, and the nanoparticles were useful to transfect N2A and HeLa cells. The cytotoxicity of 9R-chitosan/siRNA nanoparticles was tested by MTT assay and the nanoparticles showed low toxicity. 9R-chitosan/siRNA nanoparticles were considered as a potential carrier for siRNA delivery due to their low cytotoxicity and ability to transport siRNA into cells, resulting in inhibition of targeting mRNA induction.; Small interfering RNA (siRNA) has been exploited as a potential therapeutic for treatment of various diseases. However, the delivery of naked siRNA has raised several issues, including rapid enzymatic degradation, poor cellular uptake, limited stability, and low transfection efficiency. To overcome these limitations, siRNA has been combined with cationic polymers to enhance intracellular uptake and to increase stability. Chitosan is a naturally existing polysaccharide composed of glucosamine and N-acetylglucosamine residues, and can be derived by partial deacetylation of chitin obtained from crustacean shells. Chitosan is known to be biocompatible, low immunogenic, and non-toxic. Chitosan-based nanoparticles have been extensively used as a gene delivery carrier due to low toxicity and positively charged amino groups of chitosan. In this study, we hypothesized that an introduction of oligoarginine to chitosan could improve the ability of complex formation with siRNA. A peptide with nine repeating units of arginine (9R) was chemically coupled to the chitosan backbone and various characteristics of 9R-chitosan/siRNA nanoparticles were investigated. Their various physicochemical properties were characterized FT-IR, zetasizer, gel electrophoresis, FACS, and PCR. The mean diameter of 9R-chitosan/siRNA nanoparticles ranged from 210 nm to 600 nm, depending on the chitosan and 9R concentration, and the nanoparticles were useful to transfect N2A and HeLa cells. The cytotoxicity of 9R-chitosan/siRNA nanoparticles was tested by MTT assay and the nanoparticles showed low toxicity. 9R-chitosan/siRNA nanoparticles were considered as a potential carrier for siRNA delivery due to their low cytotoxicity and ability to transport siRNA into cells, resulting in inhibition of targeting mRNA induction.